The scope of the crustacean immune system for disease control

The culture or wild capture of marine and freshwater shellfish, including crustaceans, is without doubt a key source of protein for a burgeoning world population. Historically the expansion of aquaculture has, however, been accompanied by the increased incidence of economically significant diseases, most notably of viral and bacterial origin. Since the late 1970s great progress has been made in our understanding of the generalized protostome innate immune system. Distinct pathways, pathogen receptor proteins and effector molecules have since been identified that are not ancestral or homologous to those of the deuterostomes, including vertebrates. Within the past decade progress has accelerated with the rapid characterisation of new classes of recognition proteins, immune effectors and regulatory pathways. This paper provides a broad overview of our current understanding of invertebrate immunology, taking the crustacean decapod immune system as its focus. Recent developments in the field are described briefly and their implications and potential considered. These advances offer fundamental new insights in our efforts to understand disease in cultured populations and also to develop knowledge of environmental effects on host/pathogen interactions within a fishery context. Of course, challenges do remain, including the lack of an immortal cell line and the limited publically-available genomic resources. These are considered in this review as priorities for future research effort. With the continued application of more insightful technologies, coupled with associated investment, it is expected that the speed at which some of these issues are resolved will accelerate

The effect of <i>Pm</i>VRP15 gene silencing on WSSV propagation in <i>P. monodon</i> hemocytes.

<p>Transcript expression level of the WSSV genes: <i>ie-1</i>, <i>wsv477</i> and <i>vp28</i>, in <i>Pm</i>VRP15 gene-silenced <i>P. monodon</i> hemocytes were determined by qRT-PCR. Data are shown as the mean ±1 SD of three replicates and as the fold change of <i>ie-1</i>, <i>wsv477</i> and <i>vp28</i> after normalization to the EF-1α transcript levels (grey bar). The control group (GFP-dsRNA injected) are shown in the black bars.</p

The involvement of knockdown <i>Pm</i>VRP15 gene in WSSV infection in shrimp.

<p>(A) Cumulative mortality of WSSV-infected <i>Pm</i>VRP15 gene knockdown shrimp (black line) was compared with that of the control, WSSV-infected GFP gene knockdown shrimp (Grey line). Data are shown as the mean ±1 S.D. and are derived from three independent repeats. (B) After knockdown <i>Pm</i>VRP15 gene in WSSV-infected shrimp, <i>Pm</i>VRP15 gene recovery was observed after WSSV infection at 24, 36, 48 and 60 hpi.</p

Up-regulation of <i>Pm</i>VRP15 transcripts in response to WSSV infection.

<p>Relative expression ratios, as determined by qRT-PCR, of <i>Pm</i>VRP15 transcript levels in the hemocytes of WSSV-infected <i>P. monodon</i> were compared to those of the control (non-infected) shrimps and standardized against β-actin as the internal reference, at 24, 48 and 72 hpi with WSSV. The data represent the mean ±1 SD relative expression of <i>Pm</i>VRP15 post-infection (solid bar, right) and the control (open bar, left), derived from three independent experiments. Means with an asterisk are significantly different (<i>P</i><0.05, paired samples <i>t</i>-test). A relative expression ratio of <1, 1 and >1 mean that the gene expression level is down-regulated, the same or up-regulated, respectively, in the hemocytes of WSSV-infected shrimps compared to the uninfected control.</p

Western blot analysis of <i>Pm</i>VRP15 native protein in control (NaCl) and WSSV- injected (WSSV) <i>P. monodon</i> hemocytes.

<p>Hemocytes were collected at 48(HLS) was prepared and 70 μg of total HLS protein per track was subjected to duplicate SDS-PAGE resolution. Gels were then either stained with coomassie blue for total protein detection or subject to Western-blot analysis to detect <i>Pm</i>VRP15 and β-actin using specific antibodies. M is the protein size markers.</p

CFLM-derived images of the uninfected (control) and WSSV-infected hemocytes at 48 hpi with WSSV.

<p>Rabbit anti-r<i>Pm</i>VRP15 and mouse anti-VP28 primary antibodies were detected with corresponding Alexa488 and Alexa568 secondary antibodies revealing <i>Pm</i>VRP15 (green color) and VP28 (red color), respectively. Scale bars represent (A) 5 μm and (B) 2 μm. Nucleus was stained with TO-PRO-3 iodide and color was adjusted to blue. The bright field image showed hyaline cell (HC), semigranular cell (SGC) and granular cell (GC).</p

The cDNA nucleotide and deduced protein amino acid sequences of <i>Pm</i>VRP15 (GenBank accession code KF683338).

<p>The putative start codon (ATG) is in bold, the asterisk indicates the stop codon (TAA, in bold italics), the potential transmembrane domain is boxed and the proposed polyadenylation site is underlined.</p