Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: Changes in cell structure, numbers and adherence

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24 h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p < 0.01) than with 7SHRW. The cell adherence was markedly diminished (p < 0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p < 0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.     

Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis

The green-lipped mussel Perna viridis is distributed widely in the estuarine and coastal areas of the Indo-Pacific region and extensively cultured as an inexpensive protein source. Morphology and immunological activities of hemocytes of P. viridis were investigated using flow cytometry and light and electron microscopy. Three major types of hemocytes were identified in the hemolymph, including dense-granulocyte, semi-granulocyte (small and large size) and hyalinocyte. Other hemocytes, which occurred in low numbers, included granulocytes with different electron-dense/lucent granules and hemoblast-like cells. Based on flow cytometry, two subpopulations were identified. Granulocytes were larger cells, and the more abundant, containing numerous granules in the cytoplasm, and hyalinocytes were the smaller and less abundant with the fewest granules. Flow cytometry revealed that the granulocytes were more active in cell phagocytosis, contained the higher lysosomal content, and showed higher esterase activity and reactive oxygen species (ROS) generation compared with hyalinocytes. Immune functions assessed by the flow cytometry indicated that the granulocytes were the main hemocytes involved in the cellular defence in P. viridis.     

Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p = <0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.     

Feeding behavior and differential absorption of biochemical components by the infaunal bivalve Mulinia edulis and the epibenthic Mytilus chilensis in response to changes in food regimes

We measured changes in the feeding rate and food absorption efficiency of two suspension feeding bivalves, cross-trasplanted between habitats with special emphasis on their capacity for differential absorption of biochemical components from their food supply. Mulinia edulis were moved from the intertidal zone to the subtidal zone, and Mytilus chilensis from the subtidal to the intertidal zone for a period of 7 days, and then compared with animal that had not been transplanted. Experimentally prepared diets similar to those available in the two different environments were offered to the bivalves, and their rates of feeding and differential uptake of biochemical components were determined and statistically compared. The two species did not achieve complete acclimation of their feeding behaviour during the transplant period since the highest ingestion rates for biochemical components occurred under dietary conditions that reflected their habitats of origin. Absorption efficiency showed greater acclimation than the other physiological parameters measured, indicating the capacity of these species to modulate their enzymatic-digestive activity depending on food composition. We conclude that both Mytilus and Mulinia have a certain degree of physiological plasticity in their feeding behaviour and assimilatory balance of biochemical components, being greater in Mytilus. When both species encounter ambient food conditions characteristic of their normal habitats, they show maximum values of food absorption, while under conditions where their typical diets are exchanged (Mytilus in intertidal and Mulinia in subtidal), the energy absorbed declines in each, but in ways very different between the two species. Thus, Mytilus exposed to high concentrations of low quality seston reduced the energy absorbed by 31.7% compared to its normal habitat, while Mulinia exposed to low concentrations of high-quality food reduced their energy absorption by 64%.     

Giardia duodenalis: Kinetics of cyst elimination and the systemic humoral and intestinal secretory immune responses in gerbils (Meriones unguiculatus) experimentally infected

This study aimed to determine the pre-patent period and to evaluate the kinetics of cyst elimination and the systemic humoral (IgA, IgG₁, IgG_2a, IgM, IgE) and intestinal secretory (IgA) immune responses in gerbils (Meriones unguiculatus) experimentally innoculated with different doses of Giardia duodenalis trophozoites. Forty-eight animals aged 6-8 weeks were used, equally distributed among six groups, five groups innoculated with different doses of trophozoites (10¹, 10², 10³, 10⁴, 10⁵) and one control (non-infected) group. Coproparasitological examinations were carried out daily up to 91 days after inoculation (d.a.i.) to determine the pre-patent period and the kinetics of cyst elimination. Blood and stool samples were weekly collected for antibody assays. The pre-patent period was observed from the 9 d.a.i. onwards, with intermittent elimination of variable quantities of cysts up to 27 d.a.i.. All infected gerbils, irrespective of the dose received, were able to mount systemic humoral immune responses as evidenced by specific IgM titers from 7 to 28 d.a.i., corresponding to the peak of cyst elimination, followed by high and persistent IgG1 titers. Intestinal secretory responses were also seen with two peaks of fecal IgA titers, corresponding to IgM and IgG1response peaks, respectively. In conclusion, systemic and intestinal humoral immune responses were related to the control of giardiasis in this experimental model.     

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3⁺, CD4⁺, CD8⁺ and γδ T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for γδ T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.     

Stable isotope fractionation in the digestive gland, muscle and gills tissues of the marine mussel Mytilus galloprovincialis

Mytilus galloprovincialis is a common species in the Mediterranean. It is a sedentary filter-feeding organism that assimilates carbon and nitrogen isotopic ratios in tissues from its food sources. The δ¹³C and δ¹⁵N values have been used to demonstrate differences in isotopic composition between digestive gland, muscle and gills of this mussel. For δ¹³C, mean values were - 21.99 ± 0.50‰, - 19.70 ± 0.44‰, and - 19.96 ± 0.44‰, respectively; and for δ¹⁵N, they were 5.16 ± 0.90‰, 7.67 ± 0.79‰ and 7.77 ± 0.85‰, respectively. The fractionation or enrichment factor for ¹³C values between digestive gland and muscle, between digestive gland and gills, and between muscle and gills were - 2.29 ± 0.16‰, - 2.04 ± 0.14‰ and 0.27 ± 0.07‰, respectively, within the expected range of ¹³C fractionation at filter feeders reported elsewhere. In contrast, low fractionation values were found for ¹⁵N with - 2.45 ± 0.24‰, - 2.51 ± 0.16‰ and - 0.11 ± 0.16‰, between digestive gland and muscle, between digestive gland and gills, and between muscle and gills, respectively. Through isotopic fractionation of M. galloprovincialis, the depleted values were found in the digestive gland, followed by gills and then by muscle tissue. Statistical analysis (PERMANOVA) was performed to check for significant differences in δ¹³C and δ¹⁵N isotopic signatures between tissues and localities. The current study demonstrates significant differences in the δ¹³C and δ¹⁵N isotopic composition between digestive gland, muscle and gills tissues in M. galloprovincialis living in the oligotrophic environment of the Balearic Islands.     

Differences between larval and pupal hemocytes of the tobacco hornworm, Manduca sexta, determined by monoclonal antibodies and density centrifugation

Insect hemocytes play a major role in developmental processes where they disassociate and rebuild metamorphosing tissues while undergoing physiological changes themselves. We identified hemocyte changes from the last larval to the beginning of the pupal stage of the tobacco hornworm, Manduca sexta. Larval and pupal hemocytes behaved differently in a 40% Percoll density gradient. Larval granular cells were found in almost all density layers, pupal granular cells were abundant in high density layers; larval plasmatocytes occurred in dense layers, pupal plasmatocytes became enriched in less dense layers of the gradient. Using a panel of monoclonal antibodies generated against purified hemocytes, several different antibody binding patterns were identified. Quantitative differences in staining intensities were observed more often than qualitative changes, e.g. a loss or a gain of staining. Both phenomena were related to both plasmatocytes and granular cells. The distribution of the corresponding antigens in tissues was tested on cross sections of larvae and pupae as well as in Western blot analyses using organ homogenates. Several antibodies were specific for hemocytes only, among which two antibodies bound to molecules of the hematopoietic organ. Other antibodies had an additional reactivity to other tissues, mainly to the basal lamina.     

Effects of venom/calyx fluid from the endoparasitic wasp Cotesia plutellae on the hemocytes of its host Plutella xylostella in vitro

Crude venom and calyx fluid from Cotesia plutellae (Hymenoptera Braconidae) were assayed for biological activity toward hemocytes of Plutella xylostella (Lepidoptera Plutellidae). Venom from C. plutellae displayed high activity toward the spreading of plasmatocytes of P. xylostella early in the incubation period, and the inhibition was more severe as the concentration of venom increased. However, most inhibited hemocytes spread normally after being incubated for 4h. No effects were found toward granular cells from the host. Additionally, the venom from C. plutellae had some lethal effects on hemocytes of P. xylostella at high concentrations. In contrast, when incubated with different concentrations of calyx fluid, the spreading of some hemocytes was inhibited, some began to disintegrate, and some were badly damaged with only the nucleus left. After 4h, the majority of hemocytes died. The same results were observed when hemocytes were incubated in calyx fluid together with venom. These results show that calyx fluid from C. plutellae may play a major role in the suppression of the host immune system, whereas venom from C. plutellae has a limited effect on hemocytes and probably synergizes the effect of calyx fluid or polydnavirus.     

The expression of peptidoglycan recognition protein-S1 gene in the scallop Chlamys farreri was enhanced after a second challenge by Listonellaanguillarum

A more rapid and powerful response against repeated exposure of same pathogen in vertebrates is usually considered as the reflection of immunological memory, but it is not well understood in invertebrates. In the present study, the temporal expression profiles of Chlamys farreri peptidoglycan recognition protein-S1 (CfPGRP-S1) gene after two challenges of Listonella anguillarum were examined to evaluate priming response in scallops. The up-regulation of CfPGRP-S1 mRNA occurred 3 h earlier, and the expression level was significant higher (P < 0.05), after the second challenge than that after the first challenge. The preliminary results provided new insights into invertebrate immunological memory, and they also would be helpful to develop strategies for disease control.     

Sarcoptes scabiei: Specific immune response to sarcoptic mange in the Iberian ibex Capra pyrenaica depends on previous exposure and sex

Host acquired immunity is a critical factor that conditions the survival of parasites. Nevertheless, there is a shortage of data concerning inter-individual immunological inequalities in wild mammals. Sarcoptic mange is a widespread parasitosis that severely affects mammals such as the Iberian ibex (Capra pyrenaica). Despite some work on the subject, the immune response to sarcoptic mange infestation is still a complex and poorly understood phenomenon. To improve knowledge of the host-Sarcoptes immunological interaction, 18 Iberian ibexes were experimentally infested. IgG levels were assessed using ELISA to test for potential factors determining the specific immune response to infestation. Previous exposure and sex appeared to affect the IgG response to infestation and our results suggest a sex-biased immunomodulation. We discuss the immunological pattern of host-Sarcoptes interactions and also suggest further lines of work that may improve the understanding of immunological interactions of host-Sarcoptes systems.     

Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions

Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 °C for 15 min extended their life time.Galleria mellonella larvae were injected with 10⁴, 10⁵ and 10⁶ fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 10⁵ and increased when 10⁶ blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 °C for 30 min before infection extended their life time when 10³ and 10⁴ spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals.     

On the roles of Notch, Delta, kuzbanian, and inscuteable during the development of Drosophila embryonic neuroblast lineages

The generation of cellular diversity in the nervous system involves the mechanism of asymmetric cell division. Besides an array of molecules, including the Par protein cassette, a heterotrimeric G protein signalling complex, Inscuteable plays a major role in controlling asymmetric cell division, which ultimately leads to differential activation of the Notch signalling pathway and correct specification of the two daughter cells. In this context, Notch is required to be active in one sibling and inactive in the other. Here, we investigated the requirement of genes previously known to play key roles in sibling cell fate specification such as members of the Notch signalling pathway, e.g., Notch (N), Delta (Dl), and kuzbanian (kuz) and a crucial regulator of asymmetric cell division, inscuteable (insc) throughout lineage progression of 4 neuroblasts (NB1-1, MP2, NB4-2, and NB7-1). Notch-mediated cell fate specification defects were cell-autonomous and were observed in all neuroblast lineages even in cells born from late ganglion mother cells (GMC) within the lineages. We also show that Dl functions non-autonomously during NB lineage progression and clonal cells do not require Dl from within the clone. This suggests that within a NB lineage Dl is dispensable for sibling cell fate specification. Furthermore, we provide evidence that kuz is involved in sibling cell fate specification in the central nervous system. It is cell-autonomously required in the same postmitotic cells which also depend on Notch function. This indicates that KUZ is required to facilitate a functional Notch signal in the Notch-dependent cell for correct cell fate specification. Finally, we show that three neuroblast lineages (NB1-1, NB4-2, and NB7-1) require insc function for sibling cell fate specification in cells born from early GMCs whereas insc is not required in cells born from later GMCs of the same lineages. Thus, there is differential requirement for insc for cell fate specification depending on the stage of lineage progression of NBs.     

Bmpr1a is required for proper migration of the AVE through regulation of Dkk1 expression in the pre-streak mouse embryo

Here, we report a novel mechanism regulating migration of the anterior visceral endoderm (AVE) by BMP signaling through BMPRIA. In Bmpr1a-deficient (Bmpr-null) embryos, the AVE does not migrate at all. In embryos with an epiblast-specific deletion of Bmpr1a (Bmpr1a^null/flox; Sox2Cre embryos), the AVE cells migrate randomly from the distal end of embryos, resulting in an expansion of the AVE. Dkk1, which is normally expressed in the anterior proximal visceral endoderm (PxVE), is downregulated in Bmpr-null embryos, whereas it is circumferentially expressed in Bmpr1a^null/flox; Sox2Cre embryos at E5.75-6.5. These results demonstrate an association of the position of Dkk1 expressing cells with direction of the migration of AVE. In Bmpr1a^null/flox; Sox2Cre embryos, a drastic decrease of WNT signaling is observed at E6.0. Addition of WNT3A to the culture of Bmpr1a^null/flox; Sox2Cre embryos at E5.5 restores expression patterns of Dkk1 and Cer1. These data indicate that BMP signaling in the epiblast induces Wnt3 and Wnt3a expression to maintain WNT signaling in the VE, resulting in downregulation of Dkk1 to establish the anterior expression domain. Thus, our results suggest that BMP signaling regulates the expression patterns of Dkk1 for anterior migration of the AVE.     

Temporal and spatial windows delimit activation of the outer ring of wingless in the Drosophila wing

Extracellular signalling molecules play many roles in the development of higher organisms. They are used reiteratively in different tissues and stages, but the response of the receiving cells is controlled in a context dependent manner. The pattern of expression of the signalling molecule Wingless/WNT in Drosophila is extraordinarily complex. We have studied the mechanism that controls its expression and function in the outer ring of the Drosophila wing hinge. Our findings indicate that wingless expression is controlled by a dual mechanism: its initial activation requires the product of zinc finger homeodomain 2 and is subsequently repressed by the product of the gene complex elbow/no ocelli. This tight regulation restricts the activation of wingless temporally and spatially. Later in development, wingless expression is maintained by an autoregulatory loop that involves the product of homothorax. We have analyzed the phenotype of a wingless allelic combination that specifically removes the outer ring, and our results show that Wingless is required to promote local proliferation of the wing base cells. Thus, cell proliferation in the proximal-distal axis is controlled by the sequential activation of wingless in the inner ring and the outer ring at different stages of development.     

Interactive effects of food availability and aerial body temperature on the survival of two intertidal Mytilus species

Intertidal organisms must episodically contend with the rigors of both the terrestrial and the marine environments. While body temperatures during high tide are driven primarily by water temperature, aerial body temperatures are driven by multiple environmental factors such that temperature of an organism during low tide is usually quite different from air temperature. Thus, whereas decades of research have investigated the effects of water temperature on intertidal species, considerably less is known about the physiological impacts of temperature during aerial exposure at low tide, especially with regard to the interaction of aerial body temperature with other stressors. We examined the interactive effects of aerial body temperature and food supply on the survival of two intertidal blue mussels, Mytilus galloprovincialis and Mytilus trossulus. Survival was monitored for nine weeks using a simulated tidal cycle, with two levels of food and three levels of aerial body temperature (30, 25, and 20 °C). Decreased food supply significantly reduced the survival of mussels, but only under the 30 °C treatment. In the other two thermal regimes there were no significant effect of food on survival. When aerial body temperatures are high, food availability may have a greater effect on intertidal organisms. Decreases in ocean productivity have been linked to increased in ocean temperatures, thus intertidal organisms may become more susceptible to thermal stress as climates shift.