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OBJECTIVE To develop an accessible ruminant immune challenge model for rapid in vivo assessments of feed additives. ANIMALS 60 hair-breed ram lambs. METHODS Sheep were randomly assigned to 1 of 4 treatments: treatment 1, not immunosuppressed, control fed (n = 12); treatment 2, immunosuppressed, supplemented with a yeast and botanical extract (n = 18); treatment 3, immunosuppressed, supplemented with a blend of natural aluminosilicates and yeast components (n = 18); and treatment 4, immunosuppressed, control fed (n = 12). Twice-daily injections of dexamethasone (Dex; 0.1 mg/kg bodyweight, SC) were used to induce immunosuppression throughout the study (from September 25, 2020, to November 2, 2020). All sheep were immunized with keyhole limpet hemocyanin (KLH) on days 0 and 14 and injected with heataggregated KLH, ID, to induce a skin induration on day 15. Measurements included body weight (BW), average daily gain (ADG), CBC, and skin induration diameter. RESULTS Dex treatment resulted in reduced BW and ADG that was not mitigated by either feed additive. Dex reduced lymphocyte percentage, RBC count, hemoglobin, hematocrit, and skin induration diameter and increased concentrations of granulocytes and granulocyte percentage. Effects on hematocrit, hemoglobin, RBC, and skin induration diameter were mitigated with the addition of feed additives. CLINICAL RELEVANCE The described model is a tool to evaluate the ability of feed additives to mitigate the immunosuppressive effects of Dex. [ABSTRACT FROM AUTHOR]

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Ananteris is a scorpion genus that inhabits dry and seasonal areas of South and Central America. It is located in a distinctive morpho-group of Buthids, the 'Ananteris group', which also includes species distributed in the Old World. Because of the lack of information on venom composition, the study of Ananteris species could have biological and medical relevance. We conducted a venomics analysis of Ananteris platnicki, a tiny scorpion that inhabits Panama and Costa Rica, which shows the presence of putative toxins targeting ion channels, as well as proteins with similarity to hyaluronidases, proteinases, phospholipases A2, members of the CAP-domain family, and hemocyanins, among others. Venom proteolytic and hyaluronidase activities were corroborated. The determination of the primary sequences carried out by mass spectrometry evidences that several peptides are similar to the toxins present in venoms from Old World scorpion genera such as Mesobuthus, Lychas, and Isometrus, but others present in Tityus and Centruroides toxins. Even when this venom displays the characteristic protein families found in all Buthids, with a predominance of putative Na+-channel toxins and proteinases, some identified partial sequences are not common in venoms of the New World species, suggesting its differentiation into a distinctive group separated from other Buthids. [ABSTRACT FROM AUTHOR]

Conjugation to a carrier protein is essential to give rise to the antigenicity of hapten. Three carrier proteins e.g. KLH (Keyhole Limpet hemocyanin), BSA (bovine serum albumin), and OVA (Ovalbumin) were used mostly. KLH is advantageous to the others, majorly owing to its strong immunogenicity and limited usage in other biological assays. However, the cost of obtaining Keyhole Limpet is high and the solubility of KLH is not as well as the other carriers, especially after hapten conjugation. Here, we extracted the shrimp hemocyanin (SHC) from Litopenaeus vannamei (L. vannamei), which is a commonly sea product worldwide. The high pure SHC could be acquired by two-step purification, with a production yield of > 1 g proteins (98% pure) per 1 kg shrimp. Compared to KLH, the peptide-SHC conjugates exhibit higher solubility after hapten conjugation. Meanwhile, compared with KLH, SHC induces comparable antibody production efficiency in mammals, with or without conjugation. Furthermore, rabbit polyclonal antibodies or mouse monoclonal antibodies were generated by immunizing SHC-peptide conjugates, and the subsequent antibodies were confirmed to be used in western blot, immunofluorescence and immunohistochemistry. Therefore, we demonstrated that SHC may be used as a substitute for KLH in future antibody and vaccine development. [ABSTRACT FROM AUTHOR]

Odors play a key role in animal communication and mate choice. Male urinary odor becomes less attractive to mature females when males get infected or have their immune system activated. The development of the immune response at early stages is related to the production of both pro- and anti-inflammatory cytokines. To explore the chemical basis of the observed differences and analyze the effects of female interactions with the urinary odor of antigen-stimulated males, the males of inbred BALB/c (Th1) and C57BL/6 (Th2) mice were immunized with keyhole limpet hemocyanin (KLH). The odor of male urine collected 3 days after immunization proved to be less attractive to females of both strains compared to the odor of control males. Herewith, BALB/c females sniffed the urine of immunized males less frequently, while C57BL/6 females demonstrated a shorter duration of grooming in olfactory tests with urine of immunized males. The altered response of females to the odor of male urine collected on day 3 after KLH immunization matched antigen-induced modulation of relative levels of volatile organic compounds detected in urine samples by gas chromatography/mass spectroscopy (GC-MS). Males of both strains, after KLH immunization, exhibited an increase in the content of nonanal, benzothiazole, 2-sec-butyl-4,5-dihydrothiazole, and [1,4,5]-oxadithiepane. At the same time, no compounds were found, whose content changed multidirectionally in the urine of males after immunization in a strain-dependent manner. [ABSTRACT FROM AUTHOR]

Copyright of Farmacia is the property of Societatea de Stiinte Farmaceutice Romania and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This research proposal aims for the replacement of N2 molecule (instead of O2) as a final electron receptor in micro-organisms so that they can produce NH3, which is important industrially, instead of H2O. This idea is based on the hypothesis that replacement of the central atom in gas-carrying compounds in living organisms such as chlorophyll, heme and hemocyanin would replace the fixed gas. However, NH3 is an expensive product because it is produced using the Haber-Bosch reaction technique which requires high temperature and pressure. Therefore, it is predictable here to produce NH3 so simply in bioreactors which is a great economic benefit. [ABSTRACT FROM AUTHOR]

Due to the widespread use of shellfish ingredients in food products, accurate food labelling is urgently needed for consumers with shellfish allergies. Most crustacean allergen detection systems target the immunorecognition of the allergenic protein tropomyosin. However, this mode of detection may be affected by an origin-dependent protein composition. This study determined if the geographic location of capture, or aquaculture, influenced the allergenic protein profiles of Black Tiger Shrimp (Penaeus monodon), one of the most farmed and consumed shrimp species worldwide. Protein composition was analysed in shrimp from nine different locations in the Asia–Pacific by SDS-PAGE, immunoblotting, and mass spectrometry. Ten of the twelve known shrimp allergens were detected, but with considerable differences between locations. Sarcoplasmic calcium-binding protein, myosin light chain, and tropomyosin were the most abundant allergens in all locations. Hemocyanin-specific antibodies could identify up to six different isoforms, depending on the location of origin. Similarly, tropomyosin abundance varied by up to 13 times between locations. These findings suggest that allergen abundance may be related to shrimp origin and, thus, shrimp origin might directly impact the readout of commercial crustacean allergen detection kits, most of which target tropomyosin, and this should be considered in food safety assessments. [ABSTRACT FROM AUTHOR]

(1) Background: We aimed to estimate the pooled effectiveness and safety of vaccination in follicular lymphoma (FL) and discuss implications for immunotherapy development. (2) Methods: We included randomized trials (RCTs) of therapeutic vaccines in patients with FL. Progression-free survival (PFS) was the primary outcome. We searched databases (PubMed, Embase, Scopus, Web of Science Core, medRxiv) and registries (PROSPERO, CENTRAL, ClinicalTrials.gov, EuCTR, WHO ICTRP) and conducted online, citation, and manual searches. We assessed risks of bias across outcomes using RoB 2.0 and across studies using ROB-ME and a contour-enhanced funnel plot. (3) Results: Three RCTs were included (813 patients, both previously treated and untreated). Patients with a complete or partial response after chemotherapy were randomized to either a patient-specific recombinant idiotype keyhole limpet hemocyanin (Id-KLH) vaccine plus granulocyte–macrophage colony-stimulating factor (GM-CSF) or placebo immunotherapy (KLH + GM-CSF). Meta-analyses showed that PFS was worse with the vaccine, but not significantly: hazard ratio, 1.09 (95% CI 0.91–1.30). The GRADE certainty of evidence was moderate. Adverse event data were mixed. (4) Conclusions: We are moderately certain that Id-KLH results in little to no difference in PFS in FL. (5) Funding: Russian Science Foundation grant #22-25-00516. (6) Registration: PROSPERO CRD42023457528. [ABSTRACT FROM AUTHOR]

Acute ammonia exposure has detrimental effects on shrimp, but the underlying mechanisms remain to be fully explored. In the present study, we investigated the impact of acute ammonia exposure on the gut microbiota of the white shrimp Litopenaeus vannamei and its association with shrimp mortality. Exposure to a lethal concentration of ammonia for 48 h resulted in increased mortality in L. vannamei, with severe damage to the hepatopancreas. Ammonia exposure led to a significant decrease in gut microbial diversity, along with the loss of beneficial bacterial taxa and the proliferation of pathogenic Vibrio strains. A phenotypic analysis revealed a transition from the dominance of aerobic to facultative anaerobic strains due to ammonia exposure. A functional analysis revealed that ammonia exposure led to an enrichment of genes related to biofilm formation, host colonization, and virulence pathogenicity. A species-level analysis and experiments suggest the key role of a Vibrio harveyi strain in causing shrimp disease and specificity under distinct environments. These findings provide new information on the mechanism of shrimp disease under environmental changes. [ABSTRACT FROM AUTHOR]

Degraded areas are often restored through active revegetation; however, recolonization by animals is rarely engineered. Recolonization may be rapid for species with strong dispersal abilities. However, poor dispersers, such as many flightless arthropods, may struggle to recolonize newly restored sites. Actively reintroducing or "rewilding" arthropods may therefore be necessary to facilitate recolonization and restoration of arthropod communities and the ecological functions they perform. However, active interventions are rare. The purpose of this study was twofold. First, we asked whether potential source remnant arthropod communities were dispersal‐constrained and struggling to recolonize restoration sites. Second, we tested whether reintroducing entire arthropod communities from remnant populations would help dispersal‐constrained species establish during farmland ecological restoration in southern Australia. Rewilding was conducted in summer 2018 by transplanting leaf litter, soil, and entire communities contained within it from remnant source populations into geographically isolated restoration sites, which were paired with untreated controls (n = 6 remnant, rewilding transplant, and control sites). We collected leaf litter and extracted arthropod communities 19 months after the initial rewilding event, then sequenced mite, springtail, and insect communities using a metabarcoding approach. Within all groups, community similarity decreased with spatial distance between sites, suggesting significant dispersal barriers. However, only mite communities showed a strong response to rewilding, which was expressed as increased compositional similarity toward remnant sites and greater species richness relative to controls. Our results demonstrate that many arthropod species may struggle to recolonize geographically isolated restoration sites and that full community restoration requires active interventions via rewilding. [ABSTRACT FROM AUTHOR]

A marine red yeast, Rhodosporidium sphaerocarpum , is generally used for the production of lipids and carotenoids. In a previous study, we demonstrated that a marine-derived R. sphaerocarpum GDMCC 60679 can efficiently remove ammonia nitrogen and exhibit multiple probiotic functions for shrimp, Litopenaeus vannamei. Here, we performed a genome assembly of the strain GDMCC 60679 using a combination of the data from Illumina PE and PacBio CLR reads. The genome has a size of 18.03 Mb and consists of 32 contigs with an N50 length of 1,074,774 bp and GC content of 63 %. The genome was predicted to contain 6092 protein-coding genes, 5962 of which were functionally annotated. Metabolic pathways responsible for the ammonia assimilation and the synthesis of lipids and carotenoids were particularly examined to explore and characterize genes contributing to these functions. Whole-genome sequence and annotation of the strain lays a foundation to reveal the molecular mechanism of its prominent biological functions and will facilitate us to further expand new applications of yeasts in Rhodosporidium. [ABSTRACT FROM AUTHOR]

Of the many arthropod species affecting hemp (Cannabis sativa L.) cultivation in the United States, one species of particular importance is the hemp russet mite (Aculops cannabicola , HRM). Hemp russet mite is a microscopic arthropod which feeds on all parts of hemp plants. Due to its minute size, HRM can proliferate undetected for a long time, complicating management efforts and causing serious economic losses. DNA sequencing and PCR assays can facilitate accurate identification and early detection of HRM in infested-plants. Therefore, a real-time SYBR Green based species-specific PCR assay (quantitative PCR, qPCR) was developed for the identification of HRM DNA by amplification of a 104 bp Internal Transcribed Spacer 1 (ITS1) sequence. The detection limit was estimated to be approximately 48 copies of the HRM marker gene sequence. The real-time-PCR assay is rapid, detects all life stages of mite under 2 hours. A 10-fold serial dilution of the plasmid DNA containing the ITS1 insert were used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay showed a strong linear relationship with HRM DNA with R 2 of 0.96. The assay was tested against several commonly found hemp pests including two-spotted spider mite and western flower thrips to determine specificity of the assay and to show that no non-target species DNA was amplified. The outcomes of this research will have important applications for agricultural biosecurity through accurate identification of HRM, early detection and timely deployment of management tactics to manage and prevent pest outbreaks. [ABSTRACT FROM AUTHOR]

Animals synthesize simple lipids using a distinct fatty acid synthase (FAS) related to the type I polyketide synthase (PKS) enzymes that produce complex specialized metabolites. The evolutionary origin of the animal FAS and its relationship to the diversity of PKSs remain unclear despite the critical role of lipid synthesis in cellular metabolism. Recently, an animal FAS-like PKS (AFPK) was identified in sacoglossan molluscs. Here, we explore the phylogenetic distribution of AFPKs and other PKS and FAS enzymes across the tree of life. We found AFPKs widely distributed in arthropods and molluscs (>6300 newly described AFPK sequences). The AFPKs form a clade with the animal FAS, providing an evolutionary link bridging the type I PKSs and the animal FAS. We found molluscan AFPK diversification correlated with shell loss, suggesting AFPKs provide a chemical defense. Arthropods have few or no PKSs, but our results indicate AFPKs contributed to their ecological and evolutionary success by facilitating branched hydrocarbon and pheromone biosynthesis. Although animal metabolism is well studied, surprising new metabolic enzyme classes such as AFPKs await discovery. Much is still unknown of the evolution of animal metabolic enzymes. This study describes a new enzyme family bridging the production of polyketides and membrane lipids. This expands the known biochemical repertoire of animals for making ecologically and biomedically important natural products. [ABSTRACT FROM AUTHOR]

Natural products and especially those from marine organisms are being intensively explored as an alternative to synthetic antibiotics. However, the exact mechanisms of their action are not yet well understood. The molecular masses of components in the hemolymph fraction with MW 50–100 kDa from Rapana venosa were determined using ImageQuant™ TL v8.2.0 software based on electrophoretic analysis. Mainly, three types of compounds with antibacterial potential were identified, namely proteins with MW at 50.230 kDa, 62.100 kDa and 93.088 kDa that were homologous to peroxidase-like protein, aplicyanin A and L-amino acid oxidase and functional units with MW 50 kDa from R. venous hemocyanin. Data for their antibacterial effect on Escherichia coli NBIMCC 8785 were obtained by CTC/DAPI-based fluorescent analysis (analysis based on the use of a functional fluorescence probe). The fluorescent analyses demonstrated that a 50% concentration of the fraction with MW 50–100 kDa was able to eliminate 99% of the live bacteria. The antimicrobial effect was detectable even at a 1% concentration of the active compounds. The bacteria in this case had reduced metabolic activity and a 24% decreased size. The fraction had superior action compared with another mollusc product—snail slime—which killed 60% of the E. coli NBIMCC 8785 cells at a 50% concentration and had no effect at a 1% concentration. The obtained results demonstrate the high potential of the fraction with MW 50–100 kDa from R. venosa to eliminate and suppress the development of Escherichia coli NBIMCC 8785 bacteria and could be applied as an appropriate component of therapeutics with the potential to replace antibiotics to avoid the development of antibiotic resistance. [ABSTRACT FROM AUTHOR]

Bleeding of horseshoe crabs (Limulus polyphemus) for the biomedical industry can have both sublethal and lethal impacts. Bleeding induces a significant drop in the concentration of hemolymph hemocyanin, as well as decreased levels of activity. Furthermore, horseshoe crabs with low hemocyanin prior to being bled have been found to be more likely to die after the procedure. The goal of this project was to determine whether feeding horseshoe crabs after bleeding them could enhance the recovery of their hemocyanin levels and, in doing so, improve their physiological status. The feeds tested in separate experiments included (1) natural forage items, blue mussels (Mytilus edulis) or softshell clams (Mya arenaria); (2) a formulated diet containing green crabs (Carcinus maenas) and Limulus hemolymph; and (3) a modified commercially available shrimp (Litopenaeus vannamei) broodstock aquafeed. Horseshoe crabs (n = 63) were bled and then either fed or not fed, and their hemolymph hemocyanin concentrations were measured before they were bled and for the following 6–14 days. An additional 25 horseshoe crabs were treated in the same manner but not bled. In three experiments, horseshoe crabs that were fed consistently showed significantly higher hemolymph hemocyanin concentrations compared to those that were not fed. These data suggest that relatively simple modifications of the industrial bleeding procedure, such as feeding horseshoe crabs after bleeding them, may improve their physiological status prior to release. [ABSTRACT FROM AUTHOR]

Investigations of the molecular mechanisms behind detection of short, and particularly ultraviolet, wavelengths in arthropods have relied heavily on studies from insects due to the relative ease of heterologous expression of modified opsin proteins in model organisms like Drosophila. However, species outside of the Insecta can provide information on mechanisms for spectral tuning as well as the evolutionary history of pancrustacean visual pigments. Here we investigate the basis of spectral tuning in malacostracan short wavelength sensitive (SWS) opsins using phylogenetic comparative methods. Tuning sites that may be responsible for the difference between ultraviolet (UV) and violet visual pigment absorbance in the Malacostraca are identified, and the idea that an amino acid polymorphism at a single site is responsible for this shift is shown to be unlikely. Instead, we suggest that this change in absorbance is accomplished through multiple amino acid substitutions. On the basis of our findings, we conducted further surveys to identify spectral tuning mechanisms in the order Stomatopoda where duplication of UV opsins has occurred. Ancestral state reconstructions of stomatopod opsins from two main clades provide insight into the amino acid changes that lead to differing absorption by the visual pigments they form, and likely contribute the basis for the wide array of UV spectral sensitivities found in this order. [ABSTRACT FROM AUTHOR]

In the crustacean immune system, leucine-rich repeat (LRR) is one of the major structures for recognizing pathogen-associated molecular patterns (PAMPs). LRR domain-containing proteins belong to the LRR family, which is a large group of proteins with more than 6000 genes in the database. They are involved in very diverse physiological functions, mainly by interacting with other proteins. In a previous study, the LvLRRm, a transmembrane protein containing only LRR domain, was identified in the white leg shrimp, Litopenaeus vannamei. Its versatile role in performing multiple immunomodulation activities has been reported. However, there is still a lack of research on its efficient function at the protein level. To investigate its interactions with other proteins, we applied a convenient method called the 'Hybrid LRR technique' to produce a recombinant LvLRRm. The LvLRRm and hagfish's variable lymphocyte receptors (VLRs) fragments were fused to the conserved LxxLxLxxN motif while retaining the β-strand. In addition, we established interactions between hybrid proteins and the flagellin of Salmonella typhimurium by performing surface plasmon resonance (SPR) analysis. The results of the SPR analysis demonstrated notable affinity for both LvLRRm and hybrid proteins towards Salmonella flagellin. The designed LvLRRm hybrid proteins bring insight for universal applications without losing protein functions. [ABSTRACT FROM AUTHOR]

Hemocyanin is a critical oxygen-transporting protein exhibiting multiple physiological functions, including protein storage and transportation, osmolarity regulation, molting, and immunomodulation in arthropods and mollusks. However, the immune function of hemocyanin in Portunus trituberculatus is rarely reported. Here, we identified ten subunit genes within the hemocyanin family from the P. trituberculatus genome. These genes exhibited conserved functional domains and copper ion binding sites. Building upon prior research, we specifically focused on investigating the immune functions of PtHc1 and PtHc2. Tissue expression analysis revealed that PtHc1 and PtHc2 were prominently expressed in the hepatopancreas and hemocytes. Upon infection with Vibrio parahaemolyticus, both PtHc1 and PtHc2 demonstrated a similar expression pattern, characterized by a gradual upregulation of expression levels in the hepatopancreas. While within the hemocytes, they exhibited a trend of initial downregulation followed by subsequent upregulation in expression. In addition, Recombinant hemocyanin was able to inhibit bacterial proliferation in a dose-dependent manner, with inhibition rates of 40 % and 39 % when the recombinant proteins of PtHc1 and PtHc2 reached 0.03 mg/mL, respectively. Besides, V. parahaemolyticus challenge resulted in a significant 20–23 % increase in mortality in P. trituberculatus following knockdown of PtHc1 and PtHc2. Overall, our study sheds light on the immune-related functions of PtHc1 and PtHc2 in P. trituberculatus, revealing their potential as key players in defending against pathogenic threats like V. parahaemolyticus. These results enriched the field of innate immunity in crustacean and providing a theoretical basis for disease resistance breeding.

In the present study, the functional effects of hydrolyzable tannins (HT) extracted from the sweet chestnut tree Castanea sativa were evaluated either by directly incorporating them into the diet or by using a top-dressing application on the growth, body composition, total hemocyte counts, lysozyme activity, and histomorphological condition of the hepatopancreas of Pacific white shrimp Penaeus vannamei. Shrimp was confined in hapa nets installed within commercial outdoor ponds for 90 days. Eight experimental diets included a basal formulated diet (BD) with varying inclusion levels of HT (BD-0%, BD-0.1%, BD-0.2%, and BD-0.3%), a low fish meal (FM) diet with reduction on the inclusion level of FM from 10% to 7.5% and labeled as LFM 0.3% HT, a top-dressing HT application (TC) on basal diet (BDTD-0.4% HT), and a commercial diet (CDTD-0% HT and CDTD-0.4% HT). The final body weight (FBW), percentage weight gain (PWG), thermal growth coefficient (TGC), average daily growth (ADG), and feed conversion ratio (FCR) were significantly better in the group of shrimp fed with BD-0.3% HT compared to BD-0% HT. The administration of CD-0.4% HT was able to enhance the growth performance of shrimp compared to BD-0% HT and CD without HT. Higher protein and fat contents were found in the body of shrimp fed with graded levels of HT compared to shrimp fed with BD or CD without HT. Numerically, the direct inclusion and top-dressing process of HT increased the total hemocyte count and lysozyme activity in shrimp. Both BDTD-0.4% HT and CDTD-0.4% HT were also able to support a better hepatopancreatic condition with reference to histomorphology and integrity. These results indicated that BD-0.3% HT inclusion could significantly improve the growth performance and prevent the alteration in health and histomorphological condition of the hepatopancreas of shrimp P. vannamei cultured in hapa nets under commercial farm conditions, but also 0.4% HT could be used as a supplementation dosage for the top-dressing process in diets. [ABSTRACT FROM AUTHOR]

Dietary studies are essential to unravel the functioning of ecosystems and ultimately to understand biodiversity. This task, which at first may seem simple, becomes especially complex in those cases of omnivorous species with highly variable diets. In this regard, the emergence of next-generation DNA sequencing methodologies represents a powerful tool to address the problem. Here we implement a high-throughput metabarcoding strategy based on the analysis of four molecular markers aimed at sequencing both mitochondrial (animal prey) and chloroplast (diet plants) genome fragments from fecal samples of two lizard species endemic to the Balearic Archipelago (Podarcis lilfordi and P. pityusensis) obtained through non-invasive methods. The results allowed for the characterization of their diets with a high degree of taxonomic detail and have contributed a large number of new trophic records. The reported diets are based mainly on the consumption of arthropods, mollusks and plants from a diversity of taxonomic orders, as well as carrion and marine subsidies. Our analyses also reveal inter- and intra-specific differences both in terms of seasonality and geographical distribution of the sampled lizard populations. These molecular findings provide new insights into the trophic interactions of these threatened endemic lizards in their unique and isolated ecosystems. [ABSTRACT FROM AUTHOR]

The effect of exposure on the hematological parameters of the red claw crayfish Cherax quadricarinatus Von Martens 1868 has been studied. The hemolymph is taken immediately after extraction from the water after 2, 4, and 8 h of exposure on the shore and 24 and 72 h after returning to the water. The total hemocyte counts (THCs), hemogram, general protein, and hemocyanin disease are determined. The dynamics of the growth of hematological parameters in the temporal aspect is revealed: THC and the proportion of granulocytes increase to reveal the entire pattern of growth, the proportion of agranulocytes, and the total content of protein and hemocyanin. After exposure on the shore, the color of the hemolymph changes from bluish with a grayish tint to cloudy gray-green (deviation from the norm). The data indicate the development of compensatory factors and pathological properties in response to stress and the occurrence of symptoms in the air, which can be used in practice in assessing the state of the red claw crayfish in the process of transport. [ABSTRACT FROM AUTHOR]

The development of vaccine adjuvants is of interest for the management of chronic diseases, cancer, and future pandemics. Therefore, the role of Toll-like receptors (TLRs) in the effects of vaccine adjuvants has been investigated. TLR4 ligand-based adjuvants are the most frequently used adjuvants for human vaccines. Among TLR family members, TLR4 has unique dual signaling capabilities due to the recruitment of two adapter proteins, myeloid differentiation marker 88 (MyD88) and interferon-β adapter inducer containing the toll-interleukin-1 receptor (TIR) domain (TRIF). MyD88-mediated signaling triggers a proinflammatory innate immune response, while TRIF-mediated signaling leads to an adaptive immune response. Most studies have used lipopolysaccharide-based ligands as TLR4 ligand-based adjuvants; however, although protein-based ligands have been proven advantageous as adjuvants, their mechanisms of action, including their ability to undergo structural modifications to achieve optimal immunogenicity, have been explored less thoroughly. In this work, we characterized the effects of two protein-based adjuvants (PBAs) on TLR4 signaling via the recruitment of MyD88 and TRIF. As models of TLR4-PBAs, we used hemocyanin from Fissurella latimarginata (FLH) and a recombinant surface immunogenic protein (rSIP) from Streptococcus agalactiae. We determined that rSIP and FLH are partial TLR4 agonists, and depending on the protein agonist used, TLR4 has a unique bias toward the TRIF or MyD88 pathway. Furthermore, when characterizing gene products with MyD88 and TRIF pathway-dependent expression, differences in TLR4-associated signaling were observed. rSIP and FLH require MyD88 and TRIF to activate nuclear factor kappa beta (NF-kB) and interferon regulatory factor (IRF). However, rSIP and FLH have a specific pattern of interleukin 6 (IL-6) and interferon gamma-induced protein 10 (IP-10) secretion associated with MyD88 and TRIF recruitment. Functionally, rSIP and FLH promote antigen cross-presentation in a manner dependent on TLR4, MyD88 and TRIF signaling. However, FLH activates a specific TRIF-dependent signaling pathway associated with cytokine expression and a pathway dependent on MyD88 and TRIF recruitment for antigen cross-presentation. Finally, this work supports the use of these TLR4-PBAs as clinically useful vaccine adjuvants that selectively activate TRIF- and MyD88-dependent signaling to drive safe innate immune responses and vigorous Th1 adaptive immune responses. [ABSTRACT FROM AUTHOR]

The water quality including toxic gas parameters and Vibrio density is a serious problem in intensive shrimp culture. Yucca schidigera extract and Bacillus sp. are considered as a practical solution to improve the water quality. The aim of our study was to determine the effect of combination of Yucca schidigera extract and Bacillus spp. on total ammonia nitrogen (TAN) and nitrite contents, Vibrio count, and the growth performance of white leg shrimp (Penaeus vannamei). Shrimp (PL12) were assigned to seven treatments (triplicates) including treatments with combination of yucca extract and single strain of Bacillus (AY, B2Y, B3Y), combination of yucca extract and Bacillus consortium (ABBY), yucca extract only (Y), Bacillus consortium (ABB), and control treatment (C). Vibrio count was carried out by the spread plate technique every 7 days. TAN and nitrite contents were determined by using sodium nitroprusside, and sulphanilic acid with N-(1-naphthyl) ethylenediamine, respectively, at 24 hours and 168 hours after adding Bacillus strains and yucca extract. The shrimp growth parameters include the final biomass, final body weight (FBW, g), weight gain rate (WGR, %), survival rate (SR, %), and feed conversion ratio (FCR) were recorded after 5 weeks. The results showed that the water quality (0.073 – 0.179 ppm of TAN, 0.187 – 0.934 ppm of nitrite, and Vibrio count of 2.552 – 3.038 log CFU/ml) of tanks treated with combination of yucca extract and single strain of Bacillus (AY, B2Y, B3Y) or Bacillus consortium (ABBY), and Bacillus consortium (ABB) was significantly controlled compared to the control and yucca extract only treatments. The ABBY treatment most significantly improved the shrimp growth parameters (FBW, biomass, WGR, FCR, and SR - 0.424 ± 0.010 g, 40.202 ± 1.510 g, 14048.902 ± 328.756 %, 1.100 ± 0.040, and 94.667 ± 1.856 %, respectively) compared to the other treatments. Overall, our study concluded that the combination of yucca extract and Bacillus consortium could control water quality (0.036 - 0.105 ppm of TAN, 0.187 - 0.604 ppm of nitrite, and Vibrio count of 2.553 - 2.624 log CFU/ml), reduce 46.49% of FCR and significantly increase the growth performance (approximately 92.73% of FWB, 163.19% of biomass, 93.93% of WGR, 46.49% of FCR) of P. vannamei (PL12) with density of 100 individuals/100L. [ABSTRACT FROM AUTHOR]

We present a new implementation of a trust-region augmented Hessian approach (TRAH-SCF) for restricted and unrestricted Hartree–Fock and Kohn–Sham methods. With TRAH-SCF, convergence can always be achieved even with tight convergence thresholds, which requires just a modest number of iterations. Our convergence benchmark study and our illustrative applications focus on open-shell molecules, also antiferromagnetically coupled systems, for which it is notoriously complicated to converge the Roothaan–Hall self-consistent field (SCF) equations. We compare the number of TRAH iterations to reach convergence with those of Pulay's original and Kollmar's (K) variants of the direct inversion of the iterative subspace (DIIS) method and also analyze the obtained SCF solutions. Often, TRAH-SCF finds a symmetry-broken solution with a lower energy than DIIS and KDIIS. For unrestricted calculations, this is accompanied by a larger spin contamination, i.e., larger deviation from the desired spin-restricted ⟨S2⟩ expectation value. However, there are also rare cases in which DIIS finds a solution with a lower energy than KDIIS and TRAH. In rare cases, both TRAH-SCF and KDIIS may also converge to a non-aufbau solution. For those calculations, standard DIIS always diverges. For cases that converge smoothly with either method, TRAH usually needs more iterations to converge than DIIS and KDIIS because for every new set of orbitals, the level-shifted Newton–Raphson equations are solved approximately and iteratively. In such cases, the total runtime of TRAH-SCF is still competitive with the DIIS-based approaches even if extended basis sets are employed, which is illustrated for a large hemocyanin model complex. [ABSTRACT FROM AUTHOR]

The seafood industry plays a huge role in the blue economy, exploiting the advantage of the enriched protein content of marine organisms such as shrimps and molluscs, which are cultured in aquafarms. Diseases greatly affect these aquatic organisms in culture and, hence, there is need to study, in detail, their innate immune mechanisms. Hemocyanin is a non-specific innate defense molecule present in the blood cells of several invertebrates, especially molluscs, arthropods, and annelids. It is concerned with oxygen transport, blood clotting, and immune enhancement. In the present study, this macromolecular metalloprotein was isolated from the hemolymph of the marine snail Hemifusus pugilinus (Born, 1778) using Sephadex G-100 gel filtration column chromatography. It occurred as a single band (MW 80 kDa) on SDS-PAGE. High-performance liquid chromatography (HPLC) of the purified hemocyanin showed a single peak with a retention time of 4.3 min. The secondary structure and stability of the protein were detected using circular dichroism (CD), and the spectra demonstrated negative ellipticity bands close to 208 nm and 225 nm, indicating β-sheets. Further exploration of the purified hemocyanin revealed remarkable antimicrobial and antibiofilm activities against Gram-positive (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa and Proteus vulgaris) at a concentration of 1–5 μg/mL. Spectrophotometric and in situ microscopic analyses (CLSM) unveiled the potential of the purified hemocyanin to inhibit biofilm formation in these bacteria with a minimal inhibitory concentration of 40 μg/mL. Furthermore, H. pugilinus hemocyanin (10 μg/mL concentration) displayed antifungal activity against Aspergillus niger. The purified hemocyanin was also assessed for cytotoxicity against human cancer cells using cell viability assays. Altogether, the present study shows that molluscan hemocyanin is a potential antimicrobial, antibiofilm, antifungal, anticancer, and immunomodulatory agent, with great scope for application in the enhancement of the immune system of molluscs, thereby facilitating their aquaculture. [ABSTRACT FROM AUTHOR]

Simple Summary: In the adult freshwater snail, Pomacea canaliculata, their cephalic sensory tentacles can regenerate after experimental amputation. Immediately after the amputation, the wound closes and a hemocyte-rich blastema is formed, but whether hemocytes play a role in blastema formation and the regeneration process is not known. Here, we have analyzed the effects of the chemical depletion of phagocytic hemocytes on tentacle regeneration. The transient depletion of phagocytic hemocytes was achieved by injecting the snails with clodronate liposomes before tentacle amputation. Flow cytometry demonstrated the effects of clodronate liposomes on molluscan phagocytic hemocytes. Histological analysis, followed by an application of an in-house developed semi-automated hemocyte count protocol, documented that in phagocytic hemocyte-depleted snails, the regeneration process was significantly delayed. When the number of circulating phagocytic hemocytes was restored to the control values, the regeneration process recovered. The expression in the blastema of genes associated with hemocyte-mediated functions, like oxygen transport, clotting and inflammation, was evaluated using RT-qPCR. Consistent with flow cytometry and histochemical observations, the blastema from clodronate liposome-injected snails, presented significantly modified gene expression levels, thus reinforcing the hypothesis of an active role of hemocytes in the onset of tentacle regeneration. After amputation, granular hemocytes infiltrate the blastema of regenerating cephalic tentacles of the freshwater snail Pomacea canaliculata. Here, the circulating phagocytic hemocytes were chemically depleted by injecting the snails with clodronate liposomes, and the effects on the cephalic tentacle regeneration onset and on Pc-Hemocyanin, Pc-transglutaminase (Pc-TG) and Pc-Allograft Inflammatory Factor-1 (Pc-AIF-1) gene expressions were investigated. Flow cytometry analysis demonstrated that clodronate liposomes targeted large circulating hemocytes, resulting in a transient decrease in their number. Corresponding with the phagocyte depletion, tentacle regeneration onset was halted, and it resumed at the expected pace when clodronate liposome effects were no longer visible. In addition to the regeneration progress, the expressions of Pc-Hemocyanin, Pc-TG, and Pc-AIF-1, which are markers of hemocyte-mediated functions like oxygen transport and immunity, clotting, and inflammation, were modified. After the injection of clodronate liposomes, a specific computer-assisted image analysis protocol still evidenced the presence of granular hemocytes in the tentacle blastema. This is consistent with reports indicating the large and agranular hemocyte population as the most represented among the professional phagocytes of P. canaliculata and with the hypothesis that different hemocyte morphologies could exert diverse biological functions, as it has been observed in other invertebrates. [ABSTRACT FROM AUTHOR]

Hemocyanins are multimeric oxygen transport proteins present in the blood of arthropods and molluscs, containing up to 8 oxygen-binding functional units per monomer. In molluscs, hemocyanins are assembled in decamer 'building blocks' formed of 5 dimer 'plates', routinely forming didecamer or higher-order assemblies with d5 or c5 symmetry. Here we describe the cryoEM structures of the didecamer (20-mer) and tridecamer (30-mer) forms of a novel hemocyanin from the slipper limpet Crepidula fornicata (SLH) at 7.0 and 4.7 Å resolution respectively. We show that two decamers assemble in a 'tail-tail' configuration, forming a partially capped cylinder, with an additional decamer adding on in 'head-tail' configuration to make the tridecamer. Analysis of SLH samples shows substantial heterogeneity, suggesting the presence of many higher-order multimers including tetra- and pentadecamers, formed by successive addition of decamers in head-tail configuration. Retrieval of sequence data for a full-length isoform of SLH enabled the use of Alphafold to produce a molecular model of SLH, which indicated the formation of dimer slabs with high similarity to those found in keyhole limpet hemocyanin. The fit of the molecular model to the cryoEM density was excellent, showing an overall structure where the final two functional units of the subunit (FU-g and FU-h) form the partial cap at one end of the decamer, and permitting analysis of the subunit interfaces governing the assembly of tail-tail and head-tail decamer interactions as well as potential sites for N-glycosylation. Our work contributes to the understanding of higher-order oligomer formation in molluscan hemocyanins and demonstrates the utility of Alphafold for building accurate structural models of large oligomeric proteins. [ABSTRACT FROM AUTHOR]

The egg mass fluid (EMF) of the freshwater snail Biomphalaria glabrata (Hygrophila: Planorbidae) contains haemocyanin-like 1 (Hcl-1) protein, distinct from respiratory haemocyanins. The distribution of Hcl-1 was investigated among major families of Hygrophila, Physidae and Lymnaeidae, both of which employ respiratory haemocyanins, and Planorbidae, a group that evolved haemoglobin as a respiratory pigment. Immunoblotting detected c. 150 kDa protein (molecular weight of Hcl-1) cross-reactive with anti-keyhole limpet haemocyanin antiserum in the EMF of planorbids Bulinus globosus and Planorbella duryi (from a genus closely related to Biomphalaria), but not Physella acuta (Physidae) and Ladislavella elodes (Lymnaeidae). High throughput sequence data revealed Hcl-1 homologs from Bulinus globosus and Planorbella duryi, representative species that span the range of planorbid phylogeny, but not from Physella acuta (Physidae) and Lymnaea stagnalis (Lymnaeidae). A domain architecture comprising only three functional units (FUs) and predicted secondary structures within the C-terminal FU distinguish planorbid Hcl-1 protein from molluscan respiratory haemocyanins that are natively assembled as functional didecamers. Immunoblotting confirmed a monomeric configuration of native Hcl-1. Molecular clock analysis estimated divergence of Hcl-1 proteins from gastropod respiratory haemocyanins at 267 ± 143 Ma. It is hypothesized that Hcl proteins originated in the ancestor of the planorbid lineage when evolution of respiratory haemoglobin altered selective pressures for maintaining original function, facilitating mutation and refunctionalization of the ancestral respiratory haemocyanin in Planorbidae. [ABSTRACT FROM AUTHOR]

Sandy beach species are adapted to a harsh physical habitat, but for those living at relatively high latitudes, the cold, often freezing winter conditions, entail a challenge that has not been thoroughly examined. We therefore examined the individual response of talitroid amphipods (Americorchestia longicornis; Say 1818) to the strong seasonality affecting sandy beaches in Prince Edward Island, Atlantic Canada (46°N). We then experimentally assessed the influence of low temperatures upon amphipod burrowing behavior and stress levels (hemocyanin concentrations), hypothesizing that cold and freezing temperatures prompt drastic behavioral and physiological responses in the amphipods. Biweekly amphipod samples collected over three years (2019–2021) made evident the narrowness of their activity season and its tight correlation with temperature (Spearman's r = 0.85). While amphipods were abundant above 10 °C, they were very few under 5 °C and none near 0 °C. In the laboratory, amphipods exposed to 10 °C remained at shallow depths in the sand (70% at 0–4 cm of depth). By comparison, amphipods exposed to 5 °C were found at several depths (0–40 cm), while those exposed to 0 °C concentrated (70%) at depths of 16–40 cm. The assessment of stress levels showed that in comparison to amphipods at ambient temperatures, those exposed to freezing conditions exhibited hemocyanin levels 55 and 82% higher after 1 and 2 weeks, respectively. Combined, these results suggest that cold and freezing temperatures shorten the season of the amphipods and prompt them to burrow deeper into the sand and increase their stress levels, possibly influencing other life history traits. [ABSTRACT FROM AUTHOR]

Hemocyanins are oxygen-transporting glycoproteins in the hemolymph of some invertebrate species that attracted scientific interest as potential anticancer agents. The present study aims to assess the in vitro and in vivo anticancer activity of hemocyanins isolated from Helix aspersa, Helix lucorum, and Rapana venosa in the Graffi myeloid tumor model. The in vitro antitumor activity of the hemocyanins was determined by a MTT test and cytomorphological analysis by fluorescent and transmission electron microscopy. The in vivo effects of the hemocyanins were examined in hamsters transplanted with Graffi tumor. The serum antibody titers against the tested hemocyanins and tumor antigen were determined by ELISA. Histopathological assessment of the morphological features related to antitumor effect, immune system response, and toxicity in some internal organs was performed. The results of in vitro studies indicated that the tested hemocyanins induced significant antiproliferative and apoptogenic effects. The in vivo investigations demonstrated a protective antitumor effect, expressed in reduced transplantability, suppression of tumor growth and metastasis, reduced mortality, prolonged survival time, and absence of toxic side effects. The present study indicated that the antitumor activity of the studied hemocyanins was due to both immune stimulation and direct effects on the tumor cells, and they displayed their potential as therapeutic agents against hematological malignances. [ABSTRACT FROM AUTHOR]

Lectins or agglutinins are mainly proteins or glycoproteins, reported to uphold an ability to agglutinate the red blood cells (RBCs) with a known sugar specificity in a diverse group of organisms. In the present study, we purified a hemocyanin (named as MmHc) from a shrimp, Metapenaeus monoceros by size-exclusion chromatography. Further characterization revealed that the purified MmHc showed hemagglutination activity that was found to be specifically inhibited by Lewis B and Lewis Y tetrasaccharides. The MmHc displayed two oligomers of molecular weight approximately ∼78 and ∼85 kDa in SDS-PAGE. The native molecular mass of MmHc was found to be ∼457 kDa as determined by size-exclusion chromatography which indicated that the purified MmHc is an oligomeric protein. MmHc showed a maximum activity within pH 7.0–8.0, while a wide range of temperature stability was observed between 4 to 55 °C, however, it did not show any dependency on metal ions for binding. Subsequently, the analysis of the peptides by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) identified the purified MmHc as shrimp hemocyanin showing significant similarity to the hemocyanin of Penaeus vannamei. The results of multiple sequence alignment and detailed analysis of the molecular interactions predicted by AutoDock suggested that besides the oxygen carrier function, this MmHc may have multiple roles and can interact well with the Lewis Y antigen through a typical sugar binding motif containing the similar hydrophilic amino acids as the conserved residues. • A hemocyanin from a shrimp, Metapenaeus monoceros (named as MmHc), was purified by size-exclusion chromatography. • SDS- and native-PAGE analysis indicated MmHc as an oligomeric protein that was specifically inhibited by Lewis Y and B sugars. • MALDI-TOF/MS analysis revealed it as hemocyanin and showed sequence similarity with hemocyanin of shrimp. • MSA and molecular docking study revealed the presence of a typical sequence motif, whose hydrophilic amino acid residues involved in Lewis sugar interaction. [ABSTRACT FROM AUTHOR]

Genome assemblies are growing at an exponential rate and have proved indispensable for studying evolution but the effort has been biased toward vertebrates and arthropods with a particular focus on insects. Onychophora or velvet worms are an ancient group of cryptic, soil dwelling worms noted for their unique mode of prey capture, biogeographic patterns, and diversity of reproductive strategies. They constitute a poorly understood phylum of exclusively terrestrial animals that is sister group to arthropods. Due to this phylogenetic position, they are crucial in understanding the origin of the largest phylum of animals. Despite their significance, there is a paucity of genomic resources for the phylum with only one highly fragmented and incomplete genome publicly available. Initial attempts at sequencing an onychophoran genome proved difficult due to its large genome size and high repeat content. However, leveraging recent advances in long-read sequencing technology, we present here the first annotated draft genome for the phylum. With a total size of 5.6Gb, the gigantism of the Epiperipatus broadwayi genome arises from having high repeat content, intron size inflation, and extensive gene family expansion. Additionally, we report a previously unknown diversity of onychophoran hemocyanins that suggests the diversification of copper-mediated oxygen carriers occurred independently in Onychophora after its split from Arthropoda, parallel to the independent diversification of hemocyanins in each of the main arthropod lineages. [ABSTRACT FROM AUTHOR]

Experimental exposure of healthy volunteers to the T‐cell dependent neoantigen keyhole limpet hemocyanin (KLH) permits the evaluation of immunomodulatory investigational medicinal product (IMP) pharmacology prior to the recruitment of patient populations. Despite widespread use, no standardized approach to the design and conduct of such studies has been agreed. The objective of this systematic review was to survey the published literature where KLH was used as a challenge agent, describing methodology, therapeutic targets addressed, and pharmacodynamic outcome measures. We searched MEDLINE, EMBASE, clinicaltrials.gov, and Cochrane CENTRAL for studies using KLH challenge in humans between January 1, 1994, and April 1, 2022. We described key study features, including KLH formulation, dose, use of adjuvants, route of administration, co‐administered IMPs, and end points. Of 2421 titles and abstracts screened, 46 met the inclusion criteria, including 14 (31%) early phase trials of IMP, of which 10 (71%) targeted T‐cell co‐stimulation. IMPs with diverse mechanisms demonstrated modulation of the humoral response to KLH, suggesting limited specificity of this end point. Two early phase IMP studies (14%) described the response to intradermal re‐challenge (delayed type hypersensitivity). Challenge regimens for IMP assessment were often incompletely described, and exhibited marked heterogeneity, including primary KLH dose (25‐fold variation: 100–2500 mcg), KLH formulation, and co‐administration with adjuvants. Methodological heterogeneity and failure to exploit the access to tissue‐level mechanism‐relevant end points afforded by KLH challenge has impaired the translational utility of this paradigm to date. Future standardization, characterization, and methodological development is required to permit tailored, appropriately powered, mechanism‐dependent study design to optimize drug development decisions. [ABSTRACT FROM AUTHOR]

Many environmental and pathogenic insults induce endoplasmic reticulum (ER) stress in animals, especially in aquatic ecosystems, where these factors are crucial for life. In penaeid shrimp, pathogens and environmental stressors induce hemocyanin expression, but the involvement of hemocyanin in ER stress response is unknown. We demonstrate that in response to pathogenic bacteria (Vibrio parahaemolyticus and Streptococcus iniae), hemocyanin, ER stress proteins (Bip, Xbp1s, and Chop), and sterol regulatory element binding protein (SREBP) are induced to alter fatty acid levels in Penaeus vannamei. Interestingly, hemocyanin interacts with ER stress proteins to modulate SREBP expression, while ER stress inhibition with 4-Phenylbutyric acid or hemocyanin knockdown attenuates the expression of ER stress proteins, SREBP, and fatty acid levels. Contrarily, hemocyanin knockdown followed by tunicamycin treatment (ER stress activator) increased their expression. Thus, hemocyanin mediates ER stress during pathogen challenge, which consequently modulates SREBP to regulate the expression of downstream lipogenic genes and fatty acid levels. Our findings reveal a novel mechanism employed by penaeid shrimp to counteract pathogen-induced ER stress. [ABSTRACT FROM AUTHOR]

States of natural dormancy include estivation and hibernation. Ampullariids are exemplary because they undergo estivation when deprived of water or hibernation when exposed to very low temperatures. Regardless of the condition, ampullariids show increased endogenous antioxidant defenses, anticipating the expected respiratory burst during reoxygenation after reactivation, known as "Preparation for Oxidative Stress (POS)". In this work, we tested the POS hypothesis for changes in the blood and hemocytes of the bimodal breather Pomacea canaliculata (Ampullariidae) induced at experimental estivation and hibernation. We described respiratory (hemocyanin, proteins, lactate), antioxidant (GSH, uric acid, SOD, CAT, GST), and immunological (hemocyte levels, ROS production) parameters. We showed that, although the protein level remains unchanged in all experimental groups, hemocyanin increases in response to estivation. Furthermore, lactate remains unchanged in challenged snails, suggesting an aerobic metabolism during short-term challenges. Blood uric acid increases during estivation and arousal from estivation or hibernation, supporting the previously proposed antioxidant role. Regarding hemocytes, we showed that the total population increases with all challenges, and granulocytes increase during hibernation. We further showed that hibernation affects ROS production by hemocytes, possibly through mitochondrial inhibition. This study contributed to the knowledge of the adaptive strategies of ampullariids to tolerate adverse environmental conditions. [ABSTRACT FROM AUTHOR]

This study aimed at estimating genetic and phenotypic relationships among feed efficiency, immune and production traits measured pre-(9-20 weeks of age) and post-(12 weeks from on-set of lay) maturity. Production traits were average daily gain (ADG) and average daily feed-intake (ADFI1) in the pre-maturity period and age at first egg (AFE), average daily feed-intake (ADFI2) and average daily egg mass (EM) in the post-maturity period. Feed efficiency comprised of residual feed intake (RFI) estimated in both periods. Natural antibodies binding to keyhole limpet hemocyanin (KLH-IgM) and specific antibodies binding to Newcastle disease virus (NDV-IgG) measured at 16 and 28 weeks of age represented immune traits pre- and postmaturity, respectively. In the growing period, 1,820 records on ADG, KLH-IgM and NDV-IgG, and 1,559 records on ADFI1 and RFI were available for analyses. In the laying period, 1,340 records on AFE, EM, KLH-IgM and NDV-IgG, and 1,288 records on ADFI2 and RFI were used in the analyses. Bi-variate animal mixed model was fitted to estimate (co)variance components, heritability and correlations among the traits. The model constituted sex, population, generation, line and genotype as fixed effects, and animal and residual effects as random variables. During the growing period, moderate to high heritability (0.36-0.68) was estimated for the production traits and RFI while the antibody traits had low (0.10-0.22) heritability estimates. Post-maturity, the production traits and RFI were moderately (0.30-0.37) heritable while moderate to high (0.25-0.41) heritability was estimated for the antibody traits. Genetic correlations between feed efficiency and production traits in both periods showed that RFI had negative genetic correlations with ADG (-0.47) and EM (-0.56) but was positively correlated with ADFI1 (0.60), ADFI2 (0.74) and AFE (0.35). Among immune and production traits, KLH-IgM and NDV-IgG had negative genetic correlations with ADG (-0.22; -0.56), AFE (-0.39; -0.42) and EM (-0.35; -0.16) but were positively correlated with ADFI1 (0.41; 0.34) and ADFI2 (0.47; 0.52). Genetic correlations between RFI with KLH-IgM (0.62; 0.33) and NDV-IgG (0.58; 0.50) were positive in both production periods. Feed intake, RFI and antibody traits measured in both production periods were positively correlated with estimates ranging from 0.48 to 0.82. Results from this study indicate selection possibilities to improve production, feed efficiency and immune-competence in indigenous chicken. The genetic correlations suggest that improved feed efficiency would be associated with high growth rates, early maturing chicken, high egg mass and reduced feed intake. In contrast, improved general (KLH-IgM) and specific (NDV-IgG) immunity would result in lower growth rates and egg mass but associated with early sexual maturation and high feed intake. Unfavorable genetic correlations between feed efficiency and immune traits imply that chicken of higher productivity and antibody levels will consume more feed to support both functions. These associations indicate that selective breeding for feed efficiency and immune-competence may have genetic consequences on production traits and should therefore be accounted for in indigenous chicken improvement programs. [ABSTRACT FROM AUTHOR]

Under the background of climate change, increasing attention has focused on the effects of ocean deoxygenation on marine organisms. However, few studies address the effects of different food deprivation states on hypoxia tolerance. We therefore investigated the metabolic responses of the Atlantic rock crab, Cancer irroratus (starved 28–35 days, fasted 3–5 days and recently fed). Starved-crab exhibited the lowest critical oxygen saturation (Scrit), while fed-crab had the highest Scrit. The fed-crab maintained an elevated postprandial oxygen consumption (MO2) even below the Scrit of fasted-crab indicating reserved aerobic scopes for critical activities in severe hypoxia. Following feeding, hypoxia (50% and 20% oxygen saturation, SO2) retarded the specific dynamic action resulting in lower peak MO2 and longer duration. The starved-crab exhibited a lower peak MO2, prolonged duration and higher energy expenditure than fasted-crab after feeding. The decline in arterial PO2 was most pronounced below the Scrit for both fasted- and starved-crab. The higher hemocyanin concentration ([Hc]) of fasted-crab (than starved-crab) suggested they had improved oxygen transport capacity, but hypoxia did not increase [Hc] during the 72-h experiment. Following feeding, the fasted-crab significantly increased l-lactate concentration ([l-lactate]) in 20% SO2, which was not observed in starved-crab. These results suggest starvation may trigger a cross-tolerance to hypoxia. Because crabs can undergo long periods of food deprivation in their natural environment, future studies should consider how this may affect their ability to deal with environmental perturbations. [ABSTRACT FROM AUTHOR]

The bacterial disease is a major constraint to the aquaculture industry of the Chinese mitten crab (Eriocheir sinensis). Povidone-iodine (PVP-I) is a broad-spectrum chemical microbicide with fungicidal activities against pathogens. In this study, E. sinensis were soaked with PVP-I (4.38 mg/l, safe concentration) for 3 days and then infected by Aeromonas hydrophila (4 × 105 CFU/ml, 90 μl). The antioxidant capacity and immunity of E. sinensis were evaluated after being treated by PVP-I for 3 days and then infected by A. hydrophila for 3 h, 12 h, and 24 h. The results demonstrated that antioxidant capacity and immunity of E. sinensis were improved after PVP-I treatment for 3 days, and the activities of superoxide dismutase (SOD), alkaline phosphatase (AKP), and lysozyme (LZM) in hemolymph, the total hemocyte count (THC), and mRNA expression levels of Crustin in hemocytes were significantly higher than the control group. After A. hydrophila infection, E. sinensis in the PVP-I group exhibited strong antioxidant capacity. At all time points post-infection, the activities of SOD and total antioxidant capacity (T-AOC) of hemolymph in the PVP-I group were higher, and the contents of malondialdehyde (MDA) were significantly lower than in the control group. PVP-I group showed better immune performance, and the contents of THC and hemocyanin, the activities of AKP and LZM in hemolymph, and the mRNA expression of Crustin in hemocytes were usually significantly higher than the control group after infection. Therefore, short-term treatment by PVP-I can increase E. sinensis' antioxidant capacity and immunity and improve resistance to A. hydrophila. [ABSTRACT FROM AUTHOR]

Bovine serum albumin and two oxygen transport proteins, hemocyanin from the snail Achatina fulica and bovine hemoglobin, were used to define what regimens of derivative UV spectrophotometry are most appropriate for using it as an express technique for nondestructive comparative analysis of native proteins preparations. It was found that the fourth derivatives of protein absorption spectra make it possible to detect the individual bands of aromatic amino acids in a way that is optimal for solving certain practical problems. An algorithm for calculating the fourth derivatives was selected experimentally. To verify the approach, the fourth derivatives of the native proteins spectra were reconstructed by combining those of individual aromatic amino-acid spectra in the range of 240–300 nm. To demonstrate the individual differences between proteins, it is proposed to use the correlation coefficients of the fourth derivatives of spectra in the range of 240–300 nm or in the wavelength range of tyrosine and tryptophan. Although this approach does not allow estimation of the exact contents of individual aromatic amino acids in proteins, it allows comparing different proteins between each other. The proposed approach makes it possible to obtain an individual spectral "portrait" of a protein, which distinguishes it from other proteins and is useful as a reference for further experimental work with it. [ABSTRACT FROM AUTHOR]

Most current methods to assess T-cell-dependent antibody responses (TDAR) are semi-quantitative and based on measures of antibody titer generated against a standard antigen like keyhole limpet hemocyanin (KLH). The precision, sensitivity, and convenience of TDAR assays might be improved by applying rapid, sensitive, specific cytometric bead assays (CBA). In the study here, KLH antigen was covalently coupled onto the surface of cytometric beads using immune microsphere technology, and IgM antibody capture spheres were prepared for use in pretreatment processing of samples. The working parameters associated with this novel TDAR-CBA system were optimized in orthogonal experiments. The optimal concentration of the KLH coating solution in this system was 160 μg/ml, that of the anti-KLH IgG capture spheres 6.0 × 105/ml, and the optimal dilution of fluorescein isothiocyanate (FITC)-conjugated Affini-Pure Goat Anti-Mouse IgG (H + L) was 60 μg/ml. Repeated tests indicated that this approach yielded good linearity (r2 = 0.9937) method, with a within-run precision of 3.1–4.9%, and a between-run precision of 4.4–4.9%. This new approach had a limit of detection of 113.43 ng/ml (linear range = 390.63–50 000), and an interference rate of just 0.04–3.51%. Based on these findings, it seems that a new mouse TDAR assay based on CBA can be developed that would appear to be more sensitive, accurate, and precise than the current TDAR assay approaches based on traditional ELISA. [ABSTRACT FROM AUTHOR]

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Objective: Hemocyanin is a copper‐bearing protein in the hemolymph of many arthropods and mollusks and functions as an oxygen transport and important nonspecific immune protein. Methods: In this study, complementary DNA of hemocyanin isoform 2 of the prawn Macrobrachium rosenbergii (MrHc2) was isolated by rapid amplification of cDNA ends and mRNA expression was characterized to elucidate molecular basis of its function. Result: With a molecular mass of 77.3 kDa, MrHc2 contained three domains: hemocyanin‐all‐alpha, hemocyanin‐copper‐containing, and hemocyanin‐immunoglobulin‐like domains. Molecular phylogenetic analysis revealed that MrHc2 belongs to the γ‐type subunit and is closely related to hemocyanin subunit 1 of the palaemonid shrimp Macrobrachium nipponense. In addition, MrHc2 resided in a different clade relative to hemocyanin (MrHc) of M. rosenbergii (α‐type subunit) and in a different subclade relative to the hemocyanin proteins of penaeid shrimp. The messenger RNA transcript of MrHc2 was highly expressed in the hepatopancreas and weakly expressed in the gills, intestine, stomach, muscle, and hemocytes. Upon challenge with M. rosenbergii nodavirus (MrNV), the expression of MrHc2 was 1.96‐, 2.93‐, and 1.96‐fold on days 3, 4, and 5, respectively, and then gradually declined to basal levels on day 7. Conclusion: This study suggests that MrHc2 plays an important role in the innate immune response of M. rosenbergii to MrNV. Impact StatementThe study for molecular function of hemocyanin in freshwater prawn may provide strategies for the management of prawn disease especially MrNV infection. [ABSTRACT FROM AUTHOR]

Niclosamide (NI) is the main molluscicide used to control Pomacea canaliculata (Lamarck) (Architaenioglossa: Ampullariidae). However, NI failed to inhibit snail climbing during the treatment process. In this study, we examined the effect of NI combined with pedunsaponin A at an ineffective concentration. The molluscicidal effect of Pedunsaponin A on NI was evidently synergistic after 48 h, and the synergism ratio (SR) was 1.82 after treatment for 72 h at 0.8 mg·L−1. Examination of the climbing adhesion effect showed that a high concentration of Pedunsaponin A (0.4 mg·L−1 and 0.8 mg·L−1) combined with NI significantly inhibited the climbing of P. canaliculata. We further studied the synergism mechanism; the results of histopathological observation showed that the siphon appeared cavities, the muscle fibers of the ventricular were severely dissolved, and kidney tubule arrangement was distorted after NI adding Pedunsaponin A. In addition, the hemocyte survival rate and the content of hemocyanin decreased significantly. According to the results of our study, the synergism mechanism may hinder oxygen transport of P. canaliculata, influencing the supply of energy; the ability of immune defense and excretion and metabolic detoxification decreased, prolonging the action time of NI in the body. [ABSTRACT FROM AUTHOR]

Nivalenol (NIV) is a trichothecene mycotoxin that is more toxic than deoxynivalenol. It accumulates in grains due to infection with Fusarium species, which are the causative agents of scab or Fusarium head blight. An immunoassay, which is a rapid and easy analytical method, is necessary for monitoring NIV in grains. However, a specific antibody against NIV has not been prepared previously. To establish an immunoassay, we prepared NIV, introduced a linker, and generated antibodies against it. NIV was prepared from a culture of Fusarium kyushuense obtained from pressed barley through chromatographic procedures with synthetic adsorbents and silica gel. NIV was reacted with glutaric anhydride, and the reaction was stopped before mono-hemiglutaryl-NIV was changed to di-hemiglutaryl-NIV. 15-O-Hemiglutaryl-NIV was isolated via preparative HPLC and bound to keyhole limpet hemocyanin (KLH) using the active ester method. Two different monoclonal antibodies were prepared by immunizing mice with the NIV-KLH conjugate. The 50% inhibitory concentration values were 36 and 37 ng/mL. These antibodies also showed high reactivity in a direct competitive enzyme-linked immunosorbent assay and specifically reacted with NIV and 15-acetyl-NIV but not with deoxynivalenol and 4-acetyl-NIV. [ABSTRACT FROM AUTHOR]

In this study, we investigate the effect of the biofloc (BF) system using various carbon sources including sugarcane bagasse (SCB), rice bran (RB) and rice straw (RS) on growth parameters, hepatopancreatic digestive enzymes and the expression of growth‐related genes of white‐leg shrimp, Litopenaeus vannamei. Single‐nucleotide polymorphisms (SNPs) in the haemocyanin gene in small‐ and large‐sized Litopenaeus vannamei for 3 months reared in outdoor concrete ponds were also screened. The results showed that the growth and survival of shrimp in the RB and SCB groups were improved (p ≤ 0.05) and the same trend was observed for the digestive enzyme activities. The expression of different growth‐related genes including growth hormone, insulin‐like growth factor‐I and II, myosin and paramyosin was significantly increased in all biofloc groups reaching the highest levels in the RB group. The SNPs recorded in the haemocyanin gene between the small‐ and large‐sized L. vannamei revealed an insertion of Adenine at position 554 and a substitution at 1114 in large‐sized RS shrimp. So far, the RB group was attained the best growth, digestive enzyme activity and expression of growth‐related genes between different carbon sources. Furthermore, the haemocyanin gene can be used as a marker for the selection of large‐sized shrimp. [ABSTRACT FROM AUTHOR]

Epigaeic arthropods have been used worldwide as indicators of post-disturbance recovery in many different types of ecosystems. We used them to evaluate the merit of different reclamation prescriptions applied to areas disturbed by oil and gas exploration and extraction. We compared the short-term effects of different mechanical and chemical site preparation techniques on the epigaeic arthropod fauna of previously reclaimed borrow pits in arrested succession with results from plots in untreated disturbed sites and undisturbed adjacent forest. In general, arthropod diversity increased and abundance decreased with the severity of soil disturbance involved in the silvicultural prescription. We place arthropod communities into four discrete groups reflected in the treatments and the environmental characteristics of the sites: forest species, grassland species, species primarily found in herbicide plots, and species found in disturbed soil. Individual borrow pits accounted for a significant amount of variation in faunal assemblages, suggesting that site location, vagaries of colonization, or disturbance history play a significant role in how the fauna recovers post disturbance. Our study provides baseline data required to document the trajectory of recovery in these sites. Long-term monitoring is essential to evaluate the relative usefulness of reclamation prescriptions in meeting targets established by law. [ABSTRACT FROM AUTHOR]