Pulsative venous return from the branchial vessels to the heart of the bivalve Mytilus galloprovincialis supports the constant-volume mechanism.

In bivalves and gastropods, ventricle contraction causes a negative pressure in the auricles and increases venous return from the afferent oblique vein (AOV): the constant-volume (CV) mechanism. The flow in the AOV should be a pulsative flow synchronized with the ventricular contraction. The flow in the heart and adjacent vessels of Mytilus galloprovincialis were measured by magnetic resonance imaging to confirm this hypothesis. Under a regular heartbeat, pulsative flows in the AOV and branchial vessels (BVs) were almost completely synchronized with the flow in the aorta, while filling of the ventricle was in the opposite phase. Flows in the BVs were directed to the posterior direction, and a pair of BVs in the gill axes (the efferent BVs) were connected to the AOV. Based on the images of the whole pathway of the AOV in an oblique slice, we confirmed that haemolymph flow was evoked from the efferent BVs and flow into the ventricle via the auricle was completed in a single heartbeat. Therefore, the walls of the AOV and BVs could resist negative transmural pressure caused by the ventricular contraction. In conclusion, the auricle, the AOV and the BVs, including the gill filaments, act as a suction pump. The pulsative venous return is driven by the negative pressure of the AOV as in the CV mechanism, and the negative pressure in the efferent BVs could draw haemolymph from the sinus via the gill and the afferent BVs. Therefore, Mytilus can start and stop its heartbeat as necessary.

Testing of arctic insect hemolymph as a secondary agent in applied cryopreservation.

BACKGROUND: Cold hardiness of insects from extremely cold regions is based on a principle of natural cryoprotection, which is associated with physiological mechanisms provided by cryoprotectants. OBJECTIVE: Since arctic cold-hardy insects are producers of highly effective cryoprotectants, in this study, the hemolymph of Aporia crataegi L. and Upis ceramboides L. from an extremely cold area (Yakutia) was tested as a secondary component of cryoprotective agents (CPA) for cryopreservation (-80 degree C) of human peripheral blood lymphocytes and skin fibroblasts. MATERIALS AND METHODS: Lymphocytes and skin fibroblasts were treated with various combinations of DMSO and hemolymph extract and step-wise cooled to -80 degree C. Post-cryopreservation cell viability was assessed by vital staining and morphological appearance. RESULTS: Viability was higher when cells were frozen with a mixture containing DMSO and Upis ceramboides hemolymph compared to the cells frozen in DMSO, while cells frozen with DMSO and Aporia crataegi hemolymph did not survive. The fact that hemolymph of not every cold-resistant insect can be used as a secondary agent along with DMSO indicates that only a unique combination of hemolymph components and its compatibility with cells might result in a positive effect. CONCLUSION: Although the use of insect hemolymph as a complementary agent in applied cryopreservation is a problem in terms of practical application, such studies could initiate new trends in the search for the most successful hemolymph-like cryoprotectant systems. https://doi.org/10.54680/fr24210110712.

Feeding state-dependent modulation of feeding-related motor patterns.

Studies elucidating modulation of microcircuit activity in isolated nervous systems have revealed numerous insights regarding neural circuit flexibility, but this approach limits the link between experimental results and behavioral context. To bridge this gap, we studied feeding behavior-linked modulation of microcircuit activity in the isolated stomatogastric nervous system (STNS) of male Cancer borealis crabs. Specifically, we removed hemolymph from a crab that was unfed for ≥24 h ("unfed" hemolymph) or fed 15 min to 2 h before hemolymph removal ("fed" hemolymph). After feeding, the first significant foregut emptying occurred >1 h later and complete emptying required ≥6 h. We applied the unfed or fed hemolymph to the stomatogastric ganglion (STG) in an isolated STNS preparation from a separate, unfed crab to determine its influence on the VCN (ventral cardiac neuron)-triggered gastric mill (chewing) and pyloric (filtering of chewed food) rhythms. Unfed hemolymph had little influence on these rhythms, but fed hemolymph from each examined time-point (15 min, 1 h, or 2 h after feeding) slowed one or both rhythms without weakening circuit neuron activity. There were also distinct parameter changes associated with each time-point. One change unique to the 1-h time-point (i.e., reduced activity of one circuit neuron during the transition from the gastric mill retraction to protraction phase) suggested that the fed hemolymph also enhanced the influence of a projection neuron that innervates the STG from a ganglion isolated from the applied hemolymph. Hemolymph thus provides a feeding state-dependent modulation of the two feeding-related motor patterns in the C. borealis STG.NEW & NOTEWORTHY Little is known about behavior-linked modulation of microcircuit activity. We show that the VCN-triggered gastric mill (chewing) and pyloric (food filtering) rhythms in the isolated crab Cancer borealis stomatogastric nervous system were changed by applying hemolymph from recently fed but not unfed crabs. This included some distinct parameter changes during each examined post-fed hemolymph time-point. These results suggest the presence of feeding-related changes in circulating hormones that regulate consummatory microcircuit activity.

Real-time monitoring of biomechanical activity in aphids by laser speckle contrast imaging.

Studying in vivo feeding and other behaviors of small insects, such as aphids, is important for understanding their lifecycle and interaction with the environment. In this regard, the EPG (electrical penetration graph) technique is widely used to study the feeding activity in aphids. However, it is restricted to recording feeding of single insects and requires wiring insects to an electrode, impeding free movement. Hence, easy and straightforward collective observations, e.g. of groups of aphids on a plant, or probing other aphid activities in various body parts, is not possible. To circumvent these drawbacks, we developed a method based on an optical technique called laser speckle contrast imaging (LSCI). It has the potential for direct, non-invasive and contactless monitoring of a broad range of internal and external activities such as feeding, hemolymph cycling and muscle contractions in aphids or other insects. The method uses a camera and coherent light illumination of the sample. The camera records the laser speckle dynamics due to the scattering and interference of light caused by moving scatters in a probed region of the insect. Analyzing the speckle contrast allowed us to monitor and extract the activity information during aphid feeding on leaves or on artificial medium containing tracer particles. We present evidence that the observed speckle dynamics might be caused by muscle contractions, movement of hemocytes in the circulatory system or food flows in the stylets. This is the first time such a remote sensing method has been applied for optical mapping of the biomechanical activities in aphids.

A Tale of Two Transcriptomic Responses in Agricultural Pests via Host Defenses and Viral Replication.

Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) is a baculovirus that causes systemic infections in many arthropod pests. The specific molecular processes underlying the biocidal activity of AcMNPV on its insect hosts are largely unknown. We describe the transcriptional responses in two major pests, Spodoptera frugiperda (fall armyworm) and Trichoplusia ni (cabbage looper), to determine the host-pathogen responses during systemic infection, concurrently with the viral response to the host. We assembled species-specific transcriptomes of the hemolymph to identify host transcriptional responses during systemic infection and assessed the viral transcript abundance in infected hemolymph from both species. We found transcriptional suppression of chitin metabolism and tracheal development in infected hosts. Synergistic transcriptional support was observed to suggest suppression of immune responses and induction of oxidative stress indicating disease progression in the host. The entire AcMNPV core genome was expressed in the infected host hemolymph with a proportional high abundance detected for viral transcripts associated with replication, structure, and movement. Interestingly, several of the host genes that were targeted by AcMNPV as revealed by our study are also targets of chemical insecticides currently used commercially to control arthropod pests. Our results reveal an extensive overlap between biological processes represented by transcriptional responses in both hosts, as well as convergence on highly abundant viral genes expressed in the two hosts, providing an overview of the host-pathogen transcriptomic landscape during systemic infection.

The Insect Circulatory System: Structure, Function, and Evolution.

Although the insect circulatory system is involved in a multitude of vital physiological processes, it has gone grossly understudied. This review highlights this critical physiological system by detailing the structure and function of the circulatory organs, including the dorsal heart and the accessory pulsatile organs that supply hemolymph to the appendages. It also emphasizes how the circulatory system develops and ages and how, by means of reflex bleeding and functional integration with the immune system, it supports mechanisms for defense against predators and microbial invaders, respectively. Beyond that, this review details evolutionary trends and novelties associated with this system, as well as the ways in which this system also plays critical roles in thermoregulation and tracheal ventilation in high-performance fliers. Finally, this review highlights how novel discoveries could be harnessed for the control of vector-borne diseases and for translational medicine, and it details principal knowledge gaps that necessitate further investigation.

A comparison of low temperature biology of Pieris rapae from Ontario, Canada, and Yakutia, Far Eastern Russia.

Low temperatures limit the distribution and abundance of ectotherms. However, many insects can survive low temperatures by employing one of two cold tolerance strategies: freeze avoidance or freeze tolerance. Very few species can employ both strategies, but those that do provide a rare opportunity to study the mechanisms that differentiate freeze tolerance and freeze avoidance. We showed that overwintering pupae of the cabbage white butterfly Pieris rapae can be freeze tolerant or freeze avoidant. Pupae from a population of P. rapae in northeastern Russia (Yakutsk) froze at c. -9.3 °C and were freeze-tolerant in 2002-2003 when overwintered outside. However, P. rapae from both Yakutsk and southern Canada (London) acclimated to milder laboratory conditions in 2014 and 2017 froze at lower temperatures (< -20 °C) and were freeze-avoidant. Summer-collected P. rapae larvae (collected in Yakutsk in 2016) were partially freeze-tolerant, and decreased the temperature at which they froze in response to starvation at mild low temperatures (4 °C) and repeated partial freezing events. By comparing similarly-acclimated P. rapae pupae from both populations, we identified molecules that may facilitate low temperature tolerance, including the hemolymph ice-binding molecules and several potential low molecular weight cryoprotectants. Pieris rapae from Yakutsk exhibited high physiological plasticity, accumulating cryoprotectants and almost doubling their hemolymph osmolality when supercooled to -15 °C for two weeks, while the London P. rapae population exhibited minimal plasticity. We hypothesize that physiological plasticity is an important adaptation to extreme low temperatures (i.e. in Yakutsk) and may facilitate the transition between freeze avoidance and freeze tolerance.

Physiological impacts of time in holding ponds, biomedical bleeding, and recovery on the Atlantic horseshoe crab, Limulus polyphemus.

Atlantic horseshoe crabs, Limulus polyphemus (HSC), are commercially harvested along the eastern U.S. coast and bled for hemolymph used in pharmaceutical safety testing. In South Carolina, some HSCs are held in outdoor ponds before transport to facilities where they are bled and then released to the wild. This study examined whether the time HSCs are held before bleeding, bleeding itself, or the duration of the recovery after bleeding affects HSC mortality and physiological condition. Female HSCs were collected from Coffin Point Beach, South Carolina (April 22-24, 2016), held in ponds for 2, 4, 6, or 8 weeks, then bled or held as controls. Body weights, hemocyanin concentrations, and hemocyte densities were measured prior to treatment (bled/control) and at 2, 6, and 12 days of recovery. Hemocyanin concentrations declined significantly in HSCs held in ponds for 8 weeks prior to bleeding and were excluded from further analyses. Compared to some studies, HSC mortalities were low (11%). Impacts of time in holding ponds, bleeding, and recovery from bleeding on physiological measures were assessed using 3-way fixed-effects ANOVA. While duration of recovery had main effects on physiological measures, significant interactions were also present. There was an interaction of treatment and recovery duration, with control crabs having higher hemocyte densities than bled animals at days 2 and 6 of recovery. There were two significant two-way interactions influencing hemocyanin concentration: pond time and recovery, and treatment and recovery. Our study suggests both main and synergistic effects are important when assessing the physiology and mortality of HSCs harvested for biomedical purposes.

Osmoionic homeostasis in bivalve mollusks from different osmotic niches: Physiological patterns and evolutionary perspectives.

Physiological knowledge gained from questions focused on the challenges faced and strategies recruited by organisms in their habitats assumes fundamental importance about understanding the ability to survive when subjected to unfavorable situations. In the aquatic environment, salinity is particularly recognized as one of the main abiotic factors that affects the physiology of organisms. Although the physiological patterns and challenges imposed by each occupied environment are distinct, they tend to converge to osmotic oscillations. From a comparative perspective, we aimed to characterize the osmoregulatory patterns of the bivalve mollusks Corbicula largillierti (purple Asian cockle), Erodona mactroides (lagoon cockle), and Amarilladesma mactroides (white clam) - inhabitants of different osmotic niches - when submitted to hypo- and/or hyperosmotic salinity variations. We determined the hemolymph osmotic and ionic concentrations, tissue hydration, and the intracellular isosmotic regulation (IIR) from the use of osmolytes (organic and inorganic) after exposure to species-specific salinity intervals. Additionally, we incorporated phylogenetic perspectives to infer and even broaden the understanding about the patterns that comprise the osmoionic physiology of Bivalvia representatives. According to the variables analyzed in the hemolymph, the three species presented a pattern of osmoconformation. Furthermore, both ionic regulation and conformation patterns were observed in freshwater, estuarine, and marine species. The patterns verified experimentally show greater use of inorganic osmolytes compared to the participation of organic molecules, which varied according to the osmotic niche occupied in the IIR for the mantle, adductor muscle, and gills. This finding widens the classic vision about the preferential use of certain osmolytes by animals from distinct niches. Our phylogenetic perspective also indicates that environmental salinity drives physiological trait variations, including hemolymph osmolality and the ion composition of the extracellular fluid (sodium, chloride, magnesium, and calcium). We also highlight the important role played by the shared ancestry, which influences the interspecific variability of the hemolymph K+ in selected representatives of Bivalvia.

Evidence for non-colligative function of small cryoprotectants in a freeze-tolerant insect.

Freeze tolerance, the ability to survive internal ice formation, facilitates survival of some insects in cold habitats. Low-molecular-weight cryoprotectants such as sugars, polyols and amino acids are hypothesized to facilitate freeze tolerance, but their in vivo function is poorly understood. Here, we use a combination of metabolomics and manipulative experiments in vivo and ex vivo to examine the function of multiple cryoprotectants in the spring field cricket Gryllus veletis. Cold-acclimated G. veletis are freeze-tolerant and accumulate myo-inositol, proline and trehalose in their haemolymph and fat body. Injecting freeze-tolerant crickets with proline and trehalose increases survival of freezing to lower temperatures or for longer times. Similarly, exogenous myo-inositol and trehalose increase ex vivo freezing survival of fat body cells from freeze-tolerant crickets. No cryoprotectant (alone or in combination) is sufficient to confer freeze tolerance on non-acclimated, freeze-intolerant G. veletis. Given that each cryoprotectant differentially impacts survival in the frozen state, we conclude that small cryoprotectants are not interchangeable and likely function non-colligatively in insect freeze tolerance. Our study is the first to experimentally demonstrate the importance of non-colligative cryoprotectant function for insect freeze tolerance both in vivo and ex vivo, with implications for choosing new molecules for cryopreservation.

Haemolymph removal by Varroa mite destabilizes the dynamical interaction between immune effectors and virus in bees, as predicted by Volterra's model.

The association between the deformed wing virus and the parasitic mite Varroa destructor has been identified as a major cause of worldwide honeybee colony losses. The mite acts as a vector of the viral pathogen and can trigger its replication in infected bees. However, the mechanistic details underlying this tripartite interaction are still poorly defined, and, particularly, the causes of viral proliferation in mite-infested bees. Here, we develop and test a novel hypothesis that mite feeding destabilizes viral immune control through the removal of both virus and immune effectors, triggering uncontrolled viral replication. Our hypothesis is grounded on the predator-prey theory developed by Volterra, which predicts prey proliferation when both predators and preys are constantly removed from the system. Consistent with this hypothesis, we show that the experimental removal of increasing volumes of haemolymph from individual bees results in increasing viral densities. By contrast, we do not find consistent support for alternative proposed mechanisms of viral expansion via mite immune suppression or within-host viral evolution. Our results suggest that haemolymph removal plays an important role in the enhanced pathogen virulence observed in the presence of feeding Varroa mites. Overall, these results provide a new model for the mechanisms driving pathogen-parasite interactions in bees, which ultimately underpin honeybee health decline and colony losses.

The art of a hydraulic joint in a spider's leg: modelling, computational fluid dynamics (CFD) simulation, and bio-inspired design.

Important aspects of spider locomotion rely on a hydraulic mechanism. So far, this has not been theoretically analysed. In this work, the flow mechanism of a main hydraulic joint in a spider leg was studied. The purpose is to gain insight into a biohydraulic mechanism using an engineering approach to improve our understanding of the hemolymph flow path in the spider's legs and to contribute to the theoretical analysis of the spider's hydraulic transmission mechanism, thereby providing an inspiration for advanced biomimetic hydraulic systems. During the study, Micro-CT results were used to reconstruct the detailed flow channel. The high-pressure areas (inlet, joint, and closed leg end) and low pressures in between are also identified. Then, the internal flow field was investigated using computational fluid dynamics. At the same time, the method of dynamic mesh regeneration, elastic smoothing, is used to simulate muscle contraction and joint extension. The different functions of the channels are substantiated by the velocity profiles. Finally, a bionic hydraulic system was designed according to the trajectory of haemolymph in the flow channel.

The clotting system in decapod crustaceans: History, current knowledge and what we need to know beyond the models.

Hemolymph coagulation is among the major arms of the humoral immune response in crustaceans. According to the current model, hemolymph clotting in decapod crustacean relies mostly on the polymerization of the plasmatic clotting protein (CP) which is directly promoted by calcium-depended transglutaminase (TGase) released from hemocytes upon microbial stimulus or injury. However, the type of hemocytes containing TGase, and hence how the TGase is released, might vary among species. Thus, we discourse here about possible mechanisms for clotting initiation. On the other hand, the initiation of coagulation reaction in the absence of microbial elicitors is poorly understood and seems to involve hemocytes lability, yet the mechanism remains unknown. A cellular clottable protein called coagulogen, different to the plasma CP, occurs in several species and could be related with the immune response, but the biological relevance of this protein is unknown. It is also demonstrated that the clotting response is actively involved in defense against pathogens. In addition, both TGase and the CP show pleiotropic functions, and although both proteins are relatively conserved, some of their physic-chemical properties vary significantly. The occurrence of differences in the clotting system in crustaceans is conceivable given the high number of species and their diverse ecology. Results from still non-studied decapods may provide explanation for some of the issues presented here from an evolutionary perspective.

Exposure to 2,3,7,8-tetrachlorodibenzo-paradioxin (TCDD) hampers the host defense capability of a bivalve species, Tegillarca granosa.

Though increasing reports of deleterious impacts of dioxins and polychlorinated biphenyls (PCBs) on a variety of marine organisms have been described, their effects on the host defense capability of marine bivalve mollusks remain poorly understood. In the present study we used 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a representative of dioxins and PCBs to investigate its impacts on the host defense capability of the blood clam, Tegillarca granosa. After exposure of clams to a range (0, 0.01, 0.1, 1, and 10 µg/L) of TCDD for 96 h, the total count, cell type composition, and phagocytic rate of haemocytes were analyzed. In addition, alkaline phosphatase (ALP) activity, cell viability, and the extent of DNA damage of haemocytes were also investigated. Our results showed that exposure to relatively high TCDD concentrations led to significant reductions in the total count and phagocytic activity of haemocytes, which could be accounted by aggravated DNA damage and reduced cell viability. In addition, the percentage of red granulocyte was significantly decreased whereas that of basophil granulocyte was significantly increased upon high doses TCDD exposure (effective concentrations are 1 µg/L and 10 µg/L for red and basophil granulocytes, respectively). Moreover, clams exposed to TCDD had a significant higher activity of ALP, may also indicate an enhanced ability to eliminate pathogens through direct dephosphorylation process whereas a suppressed inflammatory response through indirect regulating of downstream molecular cascade reaction. These findings suggest that TCDD may hamper the host defense capability and therefore render bivalve mollusks more vulnerable to pathogen infections.

Proteome Comparisons between Hemolymph of Two Honeybee Strains (Apis mellifera ligustica) Reveal Divergent Molecular Basis in Driving Hemolymph Function and High Royal Jelly Secretion.

Hemolymph is vital for the immunity of honeybees and offers a way to investigate their physiological status. To gain novel insight into the functionality and molecular details of the hemolymph in driving increased Royal Jelly (RJ) production, we characterized and compared hemolymph proteomes across the larval and adult ages of Italian bees (ITbs) and Royal Jelly bees (RJbs), a stock selected from ITbs for increasing RJ output. Unprecedented in-depth proteome was attained with the identification of 3394 hemolymph proteins in both bee lines. The changes in proteome support the general function of hemolymph to drive development and immunity across different ages. However, age-specific proteome settings have adapted to prime the distinct physiology for larvae and adult bees. In larvae, the proteome is thought to drive temporal immunity, rapid organogenesis, and reorganization of larval structures. In adults, the proteome plays key roles in prompting tissue development and immune defense in newly emerged bees, in gland maturity in nurse bees, and in carbohydrate energy production in forager bees. Between larval and adult samples of the same age, RJbs and ITbs have tailored distinct hemolymph proteome programs to drive their physiology. In particular, in day 4 larvae and nurse bees, a large number of highly abundant proteins are enriched in protein synthesis and energy metabolism in RJbs. This implies that they have adapted their proteome to initiate different developmental trajectories and high RJ secretion in response to selection for enhanced RJ production. Our hitherto unexplored in-depth proteome coverage provides novel insight into molecular details that drive hemolymph function and high RJ production by RJbs.

A clip domain serine protease regulates the expression of proPO and hemolymph clotting in mud crab, Scylla paramamosain.

The clip domain serine proteinases (clip-SPs) play vital roles in embryonic development and in various innate immune functions in invertebrates such as antimicrobial activity, cell adhesion, hemolymph clotting, pattern recognition and regulation of the prophenoloxidase system. However, little is known about the role of the clip domain serine proteinase in Scylla paramamosain (designated SpcSP) immunity. In the present study, we cloned a clip-SP from S. paramamosain hemocytes using rapid amplification of cDNA end (RACE) approach. The full-length cDNA of SpcSP was 1823 bp, containing a 5' untranslated region (UTR) of 334 bp, an open reading frame of 1122 bp, and a 3' UTR of 367 bp. The open reading frame encoded a polypeptide of 373 amino acids with a calculated molecular weight of 39.7 kDa and an isoelectric point of 6.64. Structurally, SpcSP has a predicted 21-residue signal peptide and possessed the characteristic features of the clip domain family of serine proteases, namely one clip domain in the amino-terminal with six highly conserved cysteine residues and one enzyme active serine proteinase domain in the carboxyl-terminal with a highly conserved catalytic triad (His156, Asp226, Ser321). Phylogenetic analysis showed that SpcSP was clustered together with PtcSP (clip domain serine proteinase from Portunus trituberculatus). Quantitative real-time PCR (qPCR) analysis showed that the mRNA of SpcSP was constitutively expressed at different levels in all tested tissues in untreated S. paramamosain, with hemocytes and skin expressing the most. The transcriptional level of SpcSP in hemocytes was significantly up-regulated upon challenge with V. parahaemolyticus and LPS, indicating its involvement in antibacterial immune response. Indirect immunofluorescence analysis showed that SpcSP was expressed in the cytoplasm of all three hemocyte cell types (hyaline, semigranular and granular cells). Further, recombinant SpcSP protein exhibited strong binding ability and has antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as fungi. Moreover, knockdown of SpcSP resulted in increased hemolymph clotting time and decreased the mRNA expression of SpproPO mRNA in hemocytes. These findings therefore suggest that SpcSP plays an important role in the antimicrobial defense mechanism of S. paramamosain by regulating the expression of SpproPO and hemolymph clotting in S. paramamosain.

Proteomic profile of Ostrea edulis haemolymph in response to bonamiosis and identification of candidate proteins as resistance markers.

European flat oyster Ostrea edulis populations have suffered extensive mortalities caused by bonamiosis. The protozoan parasite Bonamia ostreae is largely responsible for this disease in Europe, while its congener B. exitiosa has been detected more recently in various European countries. Both of these intracellular parasites are able to survive and proliferate within haemocytes, the main cellular effectors of the immune system in molluscs. Two-dimensional electrophoresis was used to compare the haemolymph protein profile between Bonamia spp.-infected and non-infected oysters within 3 different stocks, a Galician stock of oysters selected for resistance against bonamiosis, a non-selected Galician stock and a selected Irish stock. Thirty-four proteins with a presumably relevant role in the oyster-Bonamia spp. interaction were identified; they were involved in major metabolic pathways, such as energy production, respiratory chain, oxidative stress, signal transduction, transcription, translation, protein degradation and cell defence. Furthermore, the haemolymph proteomic profiles of the non-infected oysters of the 2 Galician stocks were compared. As a result, 7 proteins representative of the non-infected Galician oysters selected for resistance against bonamiosis were identified; these 7 proteins could be considered as candidate markers of resistance to bonamiosis, which should be further assessed.

Screening of cold tolerance in fifteen springtail species.

Springtails (Collembola) are ubiquitous and help ecosystem processes such as the decomposition of dead plant material. Their ability to survive low winter temperatures is an important trait that partly defines their geographic distribution. The cold tolerances of 15 laboratory-reared species of springtails were investigated. Springtails were cold acclimated in the laboratory over two months in order to simulate a seasonal change in temperature during autumn. Springtails were then exposed to decreasing sub-zero temperatures and at the same time simulating the moisture conditions in frozen soil. The cold tolerance of the species reflected well the climate of region of origin. Differential scanning calorimetry of individual springtails showed that melting points of body fluids did not become lower due to long-term cold acclimation (from 20° to 1.5°C). However, both water content and melting point of two arctic species (Hypogastrura viatica and Protaphorura macfadyeni) decreased drastically during exposure to sub-zero temperatures indicating cryoprotective dehydration (CPD). These arctic species survived exposure to - 9 °C for two weeks and - 20 °C for at least one week using CPD. Four other subarctic or cool temperate species also used CPD and survived - 9 °C for weeks, whereas springtails in culture from less cool temperate regions had poor cold tolerance.

Study on establishing normal ranges of chosen biochemical parameters of haemolymph of Cornu aspersum maxima and Cepaea nemoralis gastropods.

The aim of the study was to establish normal ranges for chosen biochemical parameters of haemolymph of snails (Gasropoda: Mollusca), in the light of the use of these animals as experi- mental models in various types of studies. The study was conducted on 100 specimens of Cornu aspersum maxima (CAM) and 100 specimens of Cepaea nemoralis (CN). The haemolymph col- lected from the animals was analysed using colorimetry to assay aspartate transaminase (AST) activity, alanine transaminase (ALT) activity, amylase activity and the concentrations of urea and triglycerides. In the further part of the study, the influence of administering doxycycline with feed on the change of AST and ALT activity in snail haemolymph has been studied. The normal values established for CAM are as follow: AST activity: 26-38 u/l, ALT activity: 0-11 u/l, amylase activity 9-16 u/l, concentration of urea: 3-6 mg/dl, concentration of triglycerides: 16-20 mg/dl. For CN, the following data have been obtained: AST activity: 30-80 u/l, ALT activity: 0-15 u/l, amylase activity 12-15 u/l, concentration of urea: 5-8 mg/dl, concentration of triglycerides: 18-24 mg/dl. It has been shown that doxycycline presents a high workload on the hepatopancreas of snails, which is reflected by a statistically significant (p ⟨ 0.05) increase of AST and ALT activity in the haemolymph of the specimens which obtained doxycycline in feed, as compared to the groups with antibiotic-free feed. The haemolymph activity of both studied parameters increased together with study time and tetracycline administration time.

Biological function of unique sulfated glycosaminoglycans in primitive chordates.

Glycosaminoglycans with unique sulfation patterns have been identified in different species of ascidians (sea squirts), a group of marine invertebrates of the Phylum Chordata, sub-phylum Tunicata (or Urochordata). Oversulfated dermatan sulfate composed of [4-α-L-IdoA-(2-O-SO3)-1 â†’ 3-ß-D-GalNAc(4-OSO3)-1]n repeating disaccharide units is found in the extracellular matrix of several organs, where it seems to interact with collagen fibers. This dermatan sulfate co-localizes with a decorin-like protein, as indicated by immunohistochemical analysis. Low sulfated heparin/heparan sulfate-like glycans composed mainly of [4-α-L-IdoA-(2-OSO3)-1 â†’ 4-α-D-GlcN(SO3)-1 (6-O-SO3)-1]n and [4-α-L-IdoA-(2-O-SO3)-1 â†’ 4-α-D-GlcN(SO3)-1]n have also been described in ascidians. These heparin-like glycans occur in intracellular granules of oocyte assessory cells, named test cells, in circulating basophil-like cells in the hemolymph, and at the basement membrane of different ascidian organs. In this review, we present an overview of the structure, distribution, extracellular and intracellular localization of the sulfated glycosaminoglycans in different species and tissues of ascidians. Considering the phylogenetic position of the subphylum Tunicata in the phylum Chordata, a careful analysis of these data can reveal important information about how these glycans evolved from invertebrate to vertebrate animals.