Invertebrate Survival Journal

Distinguishing Manduca sexta haemocyte types by cytometric methods

C R von Bredow — 2022-01-22

Manduca sexta larvae possess haemocytes which are classified according to their appearance into five distinct types typical for Lepidoptera: plasmatocytes (PLs), granular cells (GRs), spherule cells (SPs), oenocytoids (OEs), and prohaemocytes (ProHCs). Some haemocytes do not fit morphologically into either class, i.e. sharing morphological criteria of two cell types, obscuring their classification. We characterised larval haemocytes of M. sexta, whose identity was confirmed by specific markers, based on morphometric and cytometric methods by light microscopy and fluorescence microscopy. For each haemocyte class, the size, the length-to-width-ratio, the nuclear morphology, the relative DNA-content and the nucleus-to-cytoplasm-ratio were measured. PLs and GRs occur in either round or spread form, the latter resulting in a high length-to-width-ratio. The shape of the nuclei also differed between haemocyte types, with almost round nuclei in GRs, ProHCs and OEs, and oval to irregularly formed nuclei in SPs and PLs. The nucleus-to-cytoplasm-ratio was shown to differ significantly between each type. PLs and OEs exhibited polyploidy, while granular cells, spherule cells and putative prohaemocytes were presumably 2n (G0/G1) or 4n (G2/M). Finally, a dichotomous guide allowing determination of the haemocyte types by means of light microscopy and DNA labelling was created.

Molecular characterization and expression ananlysis of a QM protein gene from Chinese shrimp Fenneropenaeus chinensis

L Zhang — 2022-08-18

Based on lots of results, the QM protein was proposed to be a tumor suppressor, and facilitate in the innate immune responses, especially the prophenoloxidase (proPO) activation system. In the present study, a cDNA of 761 bp for the QM of Chinese shrimp, Fenneropenaeus chinensis (designated as FcQM) was cloned via rapid amplification of cDNA ends (RACE) technique. The complete cDNA sequence of FcQM contained an open reading frame (ORF) of 663 bp, 5' untranslated regions (UTR) of 42 bp and 3' UTR of 56 bp, which encoded a polypeptide of 155 amino acid residues. The analysis of the peptide sequence of FcQM revealed that FcQM sequence contained a Ribosomal_L16 domain, an SH3-binding motif, an N-acylation site, two putative antibiotic binding sites, two putative protein kinase C phosphorylation sites and two acylamidation sites, which shared over 80 % similarity with previously identified QM protein genes. Quantitative real-time PCR (qRT-PCR) results showed that the transcript of FcQM was extensively distributed in all the tested tissues and most highly expressed in hemocytes and hepatopancreas. After challenged with Vibrio anguillarum or white spot syndrome virus (WSSV), the FcQM transcripts were significantly increased (p < 0.05) both in hepatopancreas and hemocytes. Hence, the analysis of sequence and transcription suggested that FcQM might play crucial roles responses to bacterial and viral invasion in innate immunity of arthropods. This study is the first report of the QM protein gene from F. chinensis, which would enrich the understanding of the innate immune defense mechanism in shrimp.

The isolation and identification of a pathogenic Vibrio neocaledonicus from Yesso scallop (Patinopecten yessoensis)

Z Geng — 2022-07-01

Yesso scallop (Patinopecten yessoensis) is one of the most important cultured mollusc in China. However, the diseases of Yesso scallops, especially abscess disease, occurred frequently and led to massive mortality in recent years. In the present study, 20 bacterial strains were isolated and characterized from the adductor muscle of moribund Yesso scallops in the North Yellow Sea to identify the potential pathogens, and 14 of them were grouped into genus Vibrio. Vibrio neocaledonicus (M-08 strain) was found to be the most dominant strain, and it was able to survive and exhibit haemolytic activity from 8 °C to 36 °C with the highest activity at 32 °C. The pathogenicity of M-08 in Yesso scallops was further investigated by intravalvar injection and immersion challenges, and the cumulative mortality rates were calculated to be 93 % and 53 %, respectively. The activities of superoxide dismutase (SOD) and catalase (CAT) increased after M-08 intravalvar injection, and peaked at 3 h and 6 h, respectively. The MDA contents increased and were significantly higher than that in the control group at 3 h after M-08 intravalvar injection. Immediately afterwards, immune response and haemocyte apoptosis were observed in Yesso scallops. Therefore, V. neocaledonicus is the pathogen of abscess disease in the Yesso scallops, which was able to activate the antioxidant system and immune response, and cause haemocyte apoptosis, tissue damage and death sequentially. The information would provide helpful contribution to the prevention and control of abscess disease in Yesso scallop aquaculture.

Characterization of a Novel C-type Lectin against OsHV-1 infection in Scapharca broughtonii

D Wang — 2022-03-31

As important pattern recognition receptors (PRRs), most C-type lectins (CTLs) are a class of Ca²⁺-dependent carbohydrate-binding proteins that are found to be involved in non-self-recognition and antiviral process. In this study, a new CTL, named SbCTL, was identified from ark clams, Scapharca broughtonii. The amino acid sequence of SbCTL consisted of a predicted CRD (carbohydrate recognition domain) structural domain (including 102 amino acid residues). The amino acid sequence of SbCTL shared 28 % - 39 % similarity with other CTLs. There were two potential Ca²⁺ binding sites in SbCTL. The expression of SbCTL mRNA was detected in all selected tissues, with the highest expression in the gills. Expression of SbCTL mRNA increased significantly (p < 0.05) and total vibrio content increased significantly (p <0.05) in ark clam infected with OsHV-1 at 72 h post-infection. The binding activities of recombinant SbCTL (rSbCTL) to various PAMPs with or without Ca²⁺were analyzed by ELISA, rSbCTL showed especially high binding activity to LPS in a Ca²⁺-independent manner. rSbCTL also functioned on sheltering ark clams from OsHV-1 infection in vivo, there were less mortality occurred in the rSbCTL treated group than the control. In all, it suggests that SbCTL, could served a critical role in the immune response against intruders in ark clams.

Identification of five picorna-like viruses associated with the endangered cave-dwelling bivalve Congeria kusceri (Bole, 1962)

A Scapolatiello — 2022-02-09

Congeria kusceri is a bivalve mollusk species endemic to the Dinaric Karst, which displays unique adaptations that have allowed its survival in the subterranean environment with small morphological changes compared with its fossil relatives. Anthropic activities have recently impacted the surface flow of the Neretva river, impairing the seasonal water cycle that has characterized the habitat of this species for hundreds of thousands of years. The lack of an adequate water supply, together with pollution from agricultural and farm water runoff, are posing a serious threat to C. kusceri, as evidenced by the sharp population decline observed in several locations during the past few decades.

Due to the limited knowledge available about the basic biology of this filter-feeding species, the precise factors that may affect its health status and reproduction and therefore represent a hazard for its conservation are still unclear. Here, through a transcriptomic approach, we describe the nearly-complete genomes of five C. kusceri-associated RNA viruses belonging to the Picornaviridae family and phylogenetically related with picorna-like viruses previously described in other Mollusca. Although it is presently unknown whether these viruses may have a detrimental effect on bivalve health, we observed a significant increase of viral load during the summer season.

Effect of water temperature on the behavior of Neptunea cumingii and the histology, immune enzyme activity, and transcriptome of its gills and kidneys

D Zhang — 2022-01-22

The behavior of the marine snail Neptunea cumingii cultured at different temperatures (0, 4, 8, 12, 16 (control), 20, 24, and 28 °C) and the histology, immune enzyme activity, and transcriptome of its gills and kidneys were studied using ecological and molecular methods. At 0 °C, most of the snails shrank in size and did not eat during the first 6 h. At 28 °C, snails also did not eat, death began to occur at 24 h. The histology of the gills and kidneys differed among test temperatures. At 0 °C, the morphology of the gill pieces was difficult to judge. At 24 °C, edema was present in the gill lamella, and at 28 °C the gill lamella were severely deformed. Temperature increase or decrease from 16 °C caused the columnar cells of the kidney to become shorter and more numerous. The total antioxidant capacity (T-AOC), catalase (CAT) and superoxide dismutase activities (SOD) of the gill and kidney differed significantly among the temperature conditions (p < 0.05). The DEGs were subject to GO and KEGG enrichment analysis, and which showed that most of the DEGs in gill were involved in protein folding, defolding, translation, ribosome, and most of the DEGs in kidney were involved in DNA recombination, nuclear euchromatin, RNA-directed DNA polymerase activity. Finally, the results from this study showed that N. cumingii prefers the temperature range was 8 to 16 °C.

Identification and characterization of a cyclophilin A gene from Chinese shrimp Fenneropenaeus chinensis: sequence features and expression profiles

Q Q Zhou — 2022-08-18

Cyclophilin A (CypA), a key member of the immunophilin family, is the most abundantly expressed one among the 16 identified cyclophilins. Besides acting as an intracellular receptor for cyclosporine A, CypA plays a vital role in various pathological responses such as inflammation and microbial infections. In the present study, the full-length cDNA of Fenneropenaeus chinensis CypA (FcCypA) was cloned by RACE technique. The complete sequence of FcCypA cDNA contained a 5' untranslated region (UTR) of 33 bp, a 3’ UTR of 341 bp with a polyA tail, and an open reading frame (ORF) of 495 bp encoding a polypeptide of 164 amino acids with the predicted molecular weight of 17.70 kDa. The amino acid sequence of CypA has a typical peptidyl prolyl cis-trans isomerase family tag sequence (YKGSTFHRVIPNFMCQGG) as those of other species. The mRNA transcripts of FcCypA were constitutively expressed in all the tested tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach, with the highest expression level in hepatopancreas. The mRNA expression profiles of FcCypA in hemocytes and hepatopancreas could be significantly induced by the stimulation of Vibrio anguillarum suspension and white spot syndrome virus (WSSV) stock, which verifies that CypA has an effect on viral and bacterial infection in shrimps. These results indicate that CypA may be involved in the innate immunity, biological immune response and physiological stress response of Chinese shrimps.

Host-pathogen interactions of the two native isolates of Beauveria bassiana to a predatory coccinellid, Cryptolaemous montrouzieri Mulsant (Coleoptera: Coccinellidae)

S Aghaeepour — 2022-03-31

Fungi are among the most important microorganisms affecting population dynamics of insects. Although they are used as biocontrol agents for several decades but their interactions to insect pests, predators and parasitoids are still interesting in case of virulence, host physiology and environmental persistence. Understanding the possible synergistic or antagonistic interactions of entomopathogenic fungi with other biocontrol agents mainly predators is a critical factor to achieve a successful pest control program. In the current study, effects of the two native isolates of Beauveria bassiana (AM-118 and BB3) were studied on survival, cellular immunity and antioxidant system of Cryptolaemous montrouzieri Mulsant. Bioassay results showed that both AM-118 and BB3 caused significant mortality on the third instar larvae and the adults of C. montrouzieri. Moreover, they increased total and differential hemocyte counts and significantly induced phenoloxidase activity and nodule formation at 48, 72 and 96 h post-treatment. A considerable increase was also observed in the activities of antioxidant enzymes at 72 and 96 h post-treatment. Although the isolates caused mortality on both stages, induction of immune and antioxidant systems protect C. montrouzieri against infective conidia.

XXIst scientific meeting of the Italian Association of Developmental and Comparative Immunobiology (IADCI), February 16-18 2022, Didactic Pole, Department of Biology, University of Padua, Italy

L Ballarin — 2022-05-16

4th general meeting of the COST Action 16203: STEM CELLS OF MARINE/AQUATIC INVERTEBRATES: FROM BASIC RESEARCH TO INNOVATIVE APPLICATIONS (MARISTEM), October 20, 2021, Palazzo Bo, University of Padova, ITALY

L Ballarin — 2022-03-01

Stimulation effect of probiotic bacteria Bacillus spp. and inactivated yeast on the honey bees Apis mellifera physiology and honey productivity

E Sokolova — 2022-06-15

In order to find effective and safe ways to prevent the weakening and death of honey bee colonies from various stress factors, it is necessary to focus on the stimulation of physiological processes in the bee’s body, activating their mechanisms of resistance. Bacteria Bacillus subtilis and Bacillus licheniformis produce important digestive enzymes, which have antimicrobial and detoxification effects, and also stimulate metabolic processes in the organism. The inactivated yeast Saccaromyces cerevisiae was used as a protein and vitamin component of the supplement. It was found that the addition of the studied supplement to the bees' feeding increased the activity of proteases 3.32-fold in the gut, non-specific esterases - 2.16-fold in the fat body, glutathione-S-transferases - 2.64-fold in the gut and 1.69-fold in the fat body in the Apis mellifera. Application of the supplement in the field has shown that the honey productivity per family increases 1.5-fold compared to the control.