Invertebrate Survival Journal <p>An international and open access journal published by Univeristy of Modena and Reggio Emilia devoted to prompt and innovative studies on the basic defense mechanisms in invertebrates</p> University of Modena and Reggio Emilia en-US Invertebrate Survival Journal 1824-307X Potential toxic effects of titanium dioxide nanoparticles and carbon nanotubes on land snail Helix aspersa: use of oxidative stress as a reliable biomarker for ecotoxicology assessment <p>Oxidative stress represents a main regularly informed mechanism of nanoparticles (NPs) toxicity. The present work was designed to evaluate the oxidative stress in haemolymph and digestive gland of snail <em>Helix aspersa</em> after exposure to different dermal doses: 10.8 and 2.17 µg snail<sup>-1</sup> for titanium dioxide nanoparticles (TiO<sub>2</sub>NPs), and 8.0 and 1.6 µg snail<sup>-1 </sup>for Single-Walled Carbon Nanotubes (SWCNTs) during 48 h. The results indicated that, both nanomaterials (NMs) induced significant increases in malondialdehyde (MDA) as biomarker for lipid peroxidation (LPO). However, SWCNTs induced significant decline in reduced glutathione (GSH) content compared with the control. Catalase (CAT) activity significantly increased for the all treatments greater than the control. On the other hand, activities of glutathione-<em>S</em>-transferase (GST) and glutathione peroxidase (GPx) slightly decreased for both NMs. Nanoparticles (NPs) of TiO<sub>2</sub> increased activity of glutathione reductase (GR) in both haemolymph and digestive gland homogenates, while SWCNTs treatments exhibited activities did not exceed the value of the control (0.08 U mg<sup>-1</sup> protein). The present findings indicate that, alterations of antioxidant enzymes activity and levels of MDA and GSH are recognized to oxidative stress. Consequently, the use of snail, <em>H. aspersa</em> can offer a respectable sentinel model to assess ecotoxicological effects of NMs on the gastropods.</p> K Y Abdel-Halim S R Osman H T M El-Danasoury R M Ziada Copyright (c) 2021 K Y Abdel-Halim, S R Osman, H T M El-Danasoury, R M Ziada 2021-10-07 2021-10-07 119 129 10.25431/1824-307X/isj.v18i1.119-129 Transcriptomic analysis of strain-specific and gender-specific response of silkworm to BmNPV infection <p><em>Bombyx mori</em> nucleopolyhedrovirus (BmNPV) is one of the main pathogens causing serious economic losses in sericulture. However, the molecular mechanism of silkworm resistance to BmNPV is still largely unclear, and the differences in the anti-BmNPV response between silkworms of different genders have been rarely studied. In this study, BmNPV resistant strain NB and BmNPV sensitive strain 306 of different genders were used as experimental materials to inoculate BmNPV, and their transcriptomes were sequenced to analyze their response to BmNPV. Eighteen genes specifically differentially expressed in NB after BmNPV inoculation were finally obtained through transcriptomic analysis, fourteen of which were up-regulated and four were down-regulated, suggesting that they might be related to BmNPV resistance. Among them, the expression abundance of eight genes were higher in males than in females, and one gene was in the contrary. These genes suggested that there were certain differences in the anti-BmNPV response between silkworms of different genders. This study provided a new understanding of the molecular mechanism of silkworm resistance to BmNPV and the differences in the anti-BmNPV response between silkworms of different genders, and laid a foundation for future prevention and control of BmNPV.</p> S He J Xu Y Fan F Zhu K Chen Copyright (c) 2021 S He, J Xu, Y Fan, F Zhu, K Chen 2021-08-27 2021-08-27 98 107 10.25431/1824-307X/isj.v18i1.98-107 Immunological and oxidative responses of the lesser mulberry pyralid, Glyphodes pyloalis by an aqueous extract of Artemisia annua L. <p>In this search for affordable and locally available biological substances both to farmers and environment, an aqueous extract of <em>Artemisia annua </em>L. was investigated for the first time against the lesser mulberry pyralid, <em>Glyphodes pyloalis </em>Walker a serious pest in mulberry orchards. The LC<sub>10</sub>, LC<sub>30</sub> and LC<sub>50</sub> values were estimated 12.82 %, 20.6 % and 27.35 % (W/V) respectively. The extract adversely affected oviposition, impaired immunity through reduced granulocytes and phenoloxidase activity. The increased activity of detoxifying enzymes including esterases and glutathione <em>S</em>-transferase (GST) were also observed. The enhanced antioxidant system including peroxidase (POX), catalase (CAT), glucose 6-phosphate dehydrogenase (GPDH) and superoxide dismutase (SOD) were also observed. The results of the present study may provide a very safe way to control this pest in mulberry orchard and deserve further studies.</p> Z Afraze J J Sendi Copyright (c) 2021 Z Afraze, J J Sendi 2021-06-24 2021-06-24 75 85 10.25431/1824-307X/isj.v18i1.75-85 A diet rich in diatom improves the antibacterial capacity of Pacific oyster Crassostrea gigas by enhancing norepinephrine-regulated immunomodulation <p>Microalgae such as dinoflagellate and diatom are the major food source of bivalve species, and sufficient food intake contributes to the immunity and the growth of bivalves. In the present study, a monoamine oxidase gene (named as <em>Cg</em>MAO), which is the rate-limiting enzyme of norepinephrine (NE) biosynthesis, was cloned from <em>C. gigas</em>. After the oysters were fed with a diet rich in diatom for 21 and 40 d, the NE contents in oyster serum, as well as the mRNA expression of <em>Cg</em>MAO in oyster haemocytes, increased significantly compared with control group. Besides, the mRNA expression of cytokines <em>Cg</em>TNF-1 and <em>Cg</em>IL17-5 in haemocytes and the activities of immune-related enzymes (SOD and LYZ) in oyster serum also increased significantly after diatom feeding. These results collectively suggested that sufficient microalgae intake might significantly enhance the antibacterial capacity in oyster by prompting the biosynthesis of NE and triggering the subsequent antibacterial processes modulated by NE.</p> Q Sun Y Zheng X Chen N Kong Y Wang Y Zhang Y Zong Z Liu L Wang L Song Copyright (c) 2021 Q Sun, Y Zheng, X Chen, N Kong, Y Wang, Y Zhang, Y Zong, Z Liu, L Wang, L Song 2021-04-14 2021-04-14 56 65 10.25431/1824-307X/isj.v18i1.56-65 The influence of surface waters on the bioavailability and toxicity of copper oxide nanoparticles to freshwater mussels <p>The increased commercial use of copper oxide nanoparticles (nCuO) led to the release of nanoparticles in wastewaters potentially harming the aquatic biota. The purpose of this study was to determine the toxic action of nCuO and dissolved Cu (II) to <em>Dreissena bugensis</em> freshwater mussels placed in 4 types of surface waters: aquarium, green (high conductivity), brown (high organic carbon) and 10 % municipal effluent (high conductivity and anthropogenic source of organic carbon). Mussels were exposed to 50 µg/L of nCuO or Cu (II) for 96 h at 15 °C in the above waters. The results revealed that the total Cu loadings were higher in mussels placed in organic-rich waters (brown and effluent) and exposed to either forms of Cu. Tissue Cu contents were correlated with air-time survival, lipid peroxidation, protein-ubiquitin levels and DNA strand breaks. Both surface water types and Cu forms influenced Zn (II) mobilization, glutathione S-transferase activity and protein turnover (ubiquitin binding). Based on the surface water properties, Cu (II) was more influenced by the levels and origin of the organic carbon content while nCuO was more influenced by the total suspended solids. In conclusion the toxicity of nCuO could be influenced by surface waters properties expecially when similar physiological targets are impacts by these treatments.</p> J Auclair P Turcotte C Gagnon C Gagné Copyright (c) 2021 J Auclair, P Turcotte, C Gagnon, C Gagné 2021-02-17 2021-02-17 33 45 10.25431/1824-307X/isj.v18i1.33-45 New data on C1qDC from the colonial ascidian Botryllus schlosseri <p>In the compound ascidian <em>Botryllus schlosseri</em>, we recently identified a novel C1q-domain-containing (C1qDC) protein expressed by circulating immunocytes, called BsC1qDC. It has two globular C1q domains and a signal peptide and can act either as an opsonin and facilitate the phagocytosis of nonself particles or as a cytokine and stimulate the degranulation of cytotoxic cells. In the present work, we used a commercial antibody raised against human CTRP4 (hCTRP4) to provide additional evidences of the involvement of this molecule in immune responses. The antibody was validated in immunoblot analysis and recognizes a band corresponding to the expected molecular weight inferred from the analysis of the amino acid sequence of BsC1qDC. The presence of the antibody in the culture medium in phagocytosis and degranulation assays significantly reduced the two responses.</p> <p>In addition, the relationships between complement C3 activation and <em>bsc1qdc</em> transcription was studied using the injection of C3aR agonist in the colonial vasculature.</p> A Peronato G Minervini N Franchi L Ballarin Copyright (c) 2021 A Peronato, G Minervini, N Franchi, L Ballarin 2021-10-29 2021-10-29 130 137 10.25431/1824-307X/isj.v18i1.130-137 Activity of detoxification enzymes in Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) after exposure to Beauveria bassiana (Balsamo) <p><em>Rhynchophorus ferrugineus </em>is a devastating pest of palms worldwide. An integrated management strategy largely depends on chemical insecticides but due to concerns about human health risks and environmental pollution, it’s essential to emphasize on the integrated pest management (IPM)<em>.</em> In the current research the activities of detoxification enzymes esterases (EST), alkaline phosphatases (ALP), acid phosphatases (ACP), glutathione S-transferases (GST), and acetylcholinesterase (AChE) in R. ferrugineus collected from Punjab, Baluchistan, Sindh and Khyber Pakhtunkhwa (KPK) provinces of Pakistan were estimated after infection of <em>Beauveria bassiana</em> on 3rd-, 5th- and 7th-day post-treatment. The insects were exposed by immersion method with different concentrations of <em>B. bassiana</em>. The significant increase in activities of ALP (6.09), ACP (2.51), AChE (21.28) and EST (8.61) μmol/min/mg protein was observed in KPK population, while a significant increase in the activity of GST (5.23 μmol/min/mg protein) was recorded in Baluchistan population on 7th- day. The detection of elevated activities of detoxification enzymes showed the possibility of the resistance development against <em>B. bassiana</em> in R. ferrugineus<em>.</em></p> R Ahmed S Freed A Naeem M Akmal Copyright (c) 2021 R Ahmed, S Freed, A Naeem, M Akmal 2021-09-10 2021-09-10 108 118 10.25431/1824-307X/isj.v18i1.108-118 A C-type lectin (AjSJL-1) containing DPN/WVD motifs in Apostichopus japonicus recognizes multiple microbes <p>C-type lectins (CTLs) are a superfamily of Ca<sup>2+</sup> dependent carbohydrate-recognition proteins with diversified functions ranging from embryonic development to immune defense. In the present study, a CTL containing only one CRD domain with new motifs Asp-Pro-Asn (DPN) and Trp-Val-Asp (WVD) in its Ca<sup>2+</sup> binding site 2 (designated as<em> Aj</em>SJL-1) was identified from sea cucumber <em>Apostichopus japonicus</em>. The deduced amino acid sequence of <em>Aj</em>SJL-1 was homologous to CTLs from other animals with the identities ranging from 19 % to 28.4 %. The mRNA transcripts of <em>Aj</em>SJL-1 were detected in all the examined tissues with varied abundance. The expression level of <em>Aj</em>SJL-1 mRNA in coelomocyte was up-regulated significantly at 12 h after <em>Vibrio splendidus</em> challenge. The recombinant protein of <em>Aj</em>SJL-1 (r<em>Aj</em>SJL-1) displayed significant binding activity to lipopolysaccharide, peptidoglycan, mannose and D-galactose in a Ca<sup>2+</sup>-dependent manner. Moreover, r<em>Aj</em>SJL-1 exhibited strong binding capability to <em>V. splendidus</em> but week to <em>Staphylococcus aureus</em>, <em>Bifidobacterium breve</em>,<em> Pichia pastoris </em>and <em>Yarrowia lipolytica</em> in the presence of Ca<sup>2+</sup>. These results collectively suggested that <em>Aj</em>SJL-1 with new DPN/WVD motifs served as a pattern recognition receptor in sea cucumber with the capability to bind broad-spectrum microbes and initiate the immune response against invaders.</p> Q Zhao H Wang W Wang J Li Y Liu Z Xue Z Liu L Wang L Song Copyright (c) 2021 Q Zhao, H Wang, W Wang, J Li, Y Liu, Z Xue, Z Liu, L Wang, L Song 2021-06-29 2021-06-29 86 97 10.25431/1824-307X/isj.v18i1.86-97 A Field-Friendly Loop-Mediated Isothermal Amplification (FF-LAMP) method for rapid detection of Nosema bombycis in silkworm, Bombyx mori <p>Pebrine is a destructive disease that exhibits horizontal and vertical transmission and therefore it is the only mandatory quarantine item in sericulture. Here, a field-friendly loop-mediated isothermal amplification (FF-LAMP) method has been developed and validated for the rapid detection of <em>Nosema bombycis</em>, a causative agent of pebrine disease in silkworm, <em>Bombyx mori</em>. FF-LAMP primers were selected and designed for small ribosomal subunit gene and the assay was performed to detect the <em>N. bombycis</em> infection in silkworm. The FF-LAMP reaction was effective at 6 mM MgSO<sub>4</sub>, 1.4 mM dNTPs at 63 °C. The detection range of LAMP assay was found to be 10<sup>1</sup> dilutions of <em>N. bombycis</em> spores. Specificity of the primers was tested using DNA isolated from pebrine infected silkworm, pebrine free silkworm and pure <em>N. bombycis</em> by conventional PCR and FF-LAMP assay. Results revealed that the primers were specific to <em>N. bombycis</em> DNA. The FF-LAMP assay was validated in different basic silkworm seed farms with simultaneous microscopic examination of <em>N. bombycis</em> infection. This newly developed method is highly effective, specific, sensitive and rapid in detecting <em>N. bombycis</em> infection, eliminating the DNA purification steps and usage of sophisticated equipment. This method can be used in testing large number of samples making it field friendly method in sericulture industry.</p> V Sivaprasad L Satish G Mallikarjuna N Chandrakanth A V Mary Josepha S M Moorthy Copyright (c) 2021 V Sivaprasad, L Satish, G Mallikarjuna, N Chandrakanth , A V Mary Josepha, S M Moorthy 2021-06-21 2021-06-21 66 74 10.25431/1824-307X/isj.v18i1.66-74 The temporal dynamics of bacteria in the coelomic fluid of sea cucumber Apostichopus japonicus after evisceration <p>Sea cucumbers have been emerged as important models to study organ regeneration and development owing to the capacity to regenerate its organs quickly after evisceration. Evisceration is a special defense mechanism for sea cucumber to eject all of internal organs when they encounter predators or adverse environmental conditions. However, little was known about the dynamics of bacterial community in coelomic fluid after evisceration. In the present study, evisceration was induced by intracelomic injection of 0.35 M KCl, and the significantly alternation of bacterial community in coelomic fluid of sea cucumber <em>Apostichopus japonicus</em> was observed with lower diversity and total bacterial load at 7 dpe (days post evisceration) and 14 dpe. The bacterial community was tended to restore at 28 dpe. In particular, relative abundances of Bacteroidetes and Rubritaleaceae, which involved in degradation of polysaccharides and lipid, increased significantly at 7 dpe (<em>p</em> &lt; 0.05), and returned to the original level at 28 dpe. In addition, the predicted functions of bacterial community indicated that the bacteria associated with metabolism pathways of amino acid, lipid and carbohydrate also increased significantly at 7 dpe. These results suggested that the bacterial community in coelomic fluid of <em>A.</em><em> japonicus</em> was highly dynamic and could rebuild a stable community structure after evisceration. It was suggested that the enriched metabolic related beneficial bacteria at early stage played a role after evisceration in terms of decomposing polysaccharides and lipid to provide energy.</p> C Zhang Z Yu Z Xue H Li J Zhu L Wang L Song Copyright (c) 2021 C Zhang, Z Yu, Z Xue, H Li, J Zhu, L Wang, L Song 2021-02-24 2021-02-24 46 55 10.25431/1824-307X/isj.v18i1.46-55 Evolutionary insights on a novel mussel-specific foot protein-3 gene family <p>Silky byssus threads enable a number of marine and freshwater bivalve mollusks to attach themselves to hard substrates. Byssus production is an energy-costly process, which accompany the switch from planktonic to sessile life. Pointing the attention to a small foot protein (fp-3α) first identified in<em> Perna viridis</em> and abundantly secreted during the bissogenesis, we report the presence of a fp-3α gene family in species of the <em>Mytilus </em>complex, byssogenic bivalve mollusks mostly inhabiting marine waters. In the genome of <em>Mytilus galloprovincialis </em>we identified twelve fp-3α genes showing differences in exon-intron organization and suggesting that, as in the case of arthropod and mollusk defensins, exon shuffling could have played an important role in the evolution of this gene family. Also, the different tissue expression patterns of these mussel genes support their functional diversification. All predicted fp-3α proteins curiously possess a Csαβ three-dimensional motif based on 10 highly conserved cysteines and exhibit structural similarity to invertebrate defensins. The role of these small cysteine-rich proteins in supporting the byssus-mediated mussel adhesion or their action as host defence peptides remains to be established with further study.</p> E Bortoletto P Venier A Figueras B Novoa U Rosani Copyright (c) 2021 E Bortoletto, P Venier, A Figueras , B Novoa , U Rosani 2021-01-20 2021-01-20 19 32 10.25431/1824-307X/isj.v18i1.19-32 A cold bath for a formalin-free laboratory: alternative fixative methods in early developmental stages of the sea urchin Paracentrotus lividus (Lamarck, 1816) <p>Fixatives are widespread in biological and medical research because they allow preserving specimens for a long time. Historically, formaldehyde has been the most used fixative so far, but new solutions are needed because of its carcinogenicity. In this study, we tested alternative fixative methods to find a harmless, economic, and simple-to-use methodology to fix samples for larval morphological analysis in <em>Paracentrotus lividus</em>. In two separate experiments, <em>P. lividus</em> embryos were fixed after 48 h post-fertilization by adding Formalin Free Tissue Accustain<sup>TM</sup>, NaOH-buffered Formalin Free Tissue Accustain<sup>TM</sup>, glacial ethanol and denatured ethanol at different concentrations (from 10 % to 70 %) and by submerging the vials containing the larvae in seawater at 0 °C and maintained at 4 °C for 144 h. Our results suggested that all the alternative fixatives tested do not guarantee a good quality of larvae for morphological purposes, while larvae that faced the thermal shock and were kept at 4 °C did not show any evidence of damage throughout time. The results of this study candidate this method as a good and safe substitute of formalin in studies that require morphological and taxonomic recognition and shed light on its use in other kinds of studies as well.</p> A Cannavacciuolo A Chiarore M Munari Copyright (c) 2021 A Cannavacciuolo, A Chiarore, M Munari 2021-01-15 2021-01-15 11 18 10.25431/1824-307X/isj.v18i1.11-18 A putative insulin receptor involved in immune response of Chinese mitten crab Eriocheir sinensis <p>Insulin plays important roles in metabolic homeostasis during environmental challenges. The insulin receptor is a key molecule to receive and transduce insulin signals. In the present study, a novel insulin receptor was identified from the Chinese mitten crab <em>Eriocheir sinensis</em> (designated as <em>Es</em>IR). The coding region of <em>Es</em>IR gene was 3573 bp in length and encoded 1190 amino acids with all the functional domains of mammal insulin receptors, including furin-like domain, receptor L domain, transmembrane domain, and tyrosine kinase domain. Phylogenetic analysis showed that the <em>Es</em>IR shared the closest evolutionary relationship with the insulin receptor from <em>Macrobrachium rosenbergii</em>. Cell transfection experiments confirmed that <em>Es</em>IR proteins were localized on the cytomembrane. The mRNA transcripts of <em>EsIR</em> were widely distributed in various tissues with higher abundance in hepatopancreas and eyestalk of<em> E. sinensis</em>. After <em>Aeromonas hydrophila</em> stimulation, the expression level of <em>Es</em>IR mRNA decreased from 3 h to 6 h, and then increased at 12 h. The conserved structure and subcellular localization of <em>Es</em>IR together with its sensitivity to <em>A. hydrophila</em> stimulation implied that <em>Es</em>IR was probably involved in immune response of <em>E. sinensis.</em> The present study provided clues for the further investigation about the evolution and function of the insulin signaling pathway in invertebrates.</p> L Wang H Chen L Qiu L Wang L Song Copyright (c) 2021 L Wang , H Chen , L Qiu , L Wang, L Song 2021-01-14 2021-01-14 1 10 10.25431/1824-307X/isj.v18i1.1-10