Invertebrate Survival Journal

A Field-Friendly Loop-Mediated Isothermal Amplification (FF-LAMP) method for rapid detection of Nosema bombycis in silkworm, Bombyx mori

V Sivaprasad — 2021-06-21

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 Nosema bombycis, a causative agent of pebrine disease in silkworm, Bombyx mori. FF-LAMP primers were selected and designed for small ribosomal subunit gene and the assay was performed to detect the N. bombycis infection in silkworm. The FF-LAMP reaction was effective at 6 mM MgSO₄, 1.4 mM dNTPs at 63 °C. The detection range of LAMP assay was found to be 10¹ dilutions of N. bombycis spores. Specificity of the primers was tested using DNA isolated from pebrine infected silkworm, pebrine free silkworm and pure N. bombycis by conventional PCR and FF-LAMP assay. Results revealed that the primers were specific to N. bombycis DNA. The FF-LAMP assay was validated in different basic silkworm seed farms with simultaneous microscopic examination of N. bombycis infection. This newly developed method is highly effective, specific, sensitive and rapid in detecting N. bombycis 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.

The temporal dynamics of bacteria in the coelomic fluid of sea cucumber Apostichopus japonicus after evisceration

C Zhang — 2021-02-24

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 Apostichopus japonicus 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 (p < 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 A. japonicus 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.

Immunological and oxidative responses of the lesser mulberry pyralid, Glyphodes pyloalis by an aqueous extract of Artemisia annua L.

Z Afraze — 2021-06-24

In this search for affordable and locally available biological substances both to farmers and environment, an aqueous extract of Artemisia annua L. was investigated for the first time against the lesser mulberry pyralid, Glyphodes pyloalis Walker a serious pest in mulberry orchards. The LC₁₀, LC₃₀ and LC₅₀ 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 S-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.

A diet rich in diatom improves the antibacterial capacity of Pacific oyster Crassostrea gigas by enhancing norepinephrine-regulated immunomodulation

Q Sun — 2021-04-14

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 CgMAO), which is the rate-limiting enzyme of norepinephrine (NE) biosynthesis, was cloned from C. gigas. 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 CgMAO in oyster haemocytes, increased significantly compared with control group. Besides, the mRNA expression of cytokines CgTNF-1 and CgIL17-5 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.

The influence of surface waters on the bioavailability and toxicity of copper oxide nanoparticles to freshwater mussels

J Auclair — 2021-02-17

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 Dreissena bugensis 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.

A C-type lectin (AjSJL-1) containing DPN/WVD motifs in Apostichopus japonicus recognizes multiple microbes

Q Zhao — 2021-06-29

C-type lectins (CTLs) are a superfamily of Ca²⁺ 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 Ca²⁺ binding site 2 (designated as AjSJL-1) was identified from sea cucumber Apostichopus japonicus. The deduced amino acid sequence of AjSJL-1 was homologous to CTLs from other animals with the identities ranging from 19 % to 28.4 %. The mRNA transcripts of AjSJL-1 were detected in all the examined tissues with varied abundance. The expression level of AjSJL-1 mRNA in coelomocyte was up-regulated significantly at 12 h after Vibrio splendidus challenge. The recombinant protein of AjSJL-1 (rAjSJL-1) displayed significant binding activity to lipopolysaccharide, peptidoglycan, mannose and D-galactose in a Ca²⁺-dependent manner. Moreover, rAjSJL-1 exhibited strong binding capability to V. splendidus but week to Staphylococcus aureus, Bifidobacterium breve, Pichia pastoris and Yarrowia lipolytica in the presence of Ca²⁺. These results collectively suggested that AjSJL-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.

Evolutionary insights on a novel mussel-specific foot protein-3 gene family

E Bortoletto — 2021-01-20

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 Perna viridis and abundantly secreted during the bissogenesis, we report the presence of a fp-3α gene family in species of the Mytilus complex, byssogenic bivalve mollusks mostly inhabiting marine waters. In the genome of Mytilus galloprovincialis 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.

A cold bath for a formalin-free laboratory: alternative fixative methods in early developmental stages of the sea urchin Paracentrotus lividus (Lamarck, 1816)

A Cannavacciuolo — 2021-01-15

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 Paracentrotus lividus. In two separate experiments, P. lividus embryos were fixed after 48 h post-fertilization by adding Formalin Free Tissue Accustain^TM, NaOH-buffered Formalin Free Tissue Accustain^TM, 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.

A putative insulin receptor involved in immune response of Chinese mitten crab Eriocheir sinensis

L Wang — 2021-01-14

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 Eriocheir sinensis (designated as EsIR). The coding region of EsIR 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 EsIR shared the closest evolutionary relationship with the insulin receptor from Macrobrachium rosenbergii. Cell transfection experiments confirmed that EsIR proteins were localized on the cytomembrane. The mRNA transcripts of EsIR were widely distributed in various tissues with higher abundance in hepatopancreas and eyestalk of E. sinensis. After Aeromonas hydrophila stimulation, the expression level of EsIR mRNA decreased from 3 h to 6 h, and then increased at 12 h. The conserved structure and subcellular localization of EsIR together with its sensitivity to A. hydrophila stimulation implied that EsIR was probably involved in immune response of E. sinensis. The present study provided clues for the further investigation about the evolution and function of the insulin signaling pathway in invertebrates.