OPEN ACCESS of Pacific white shrimp

Overview

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Key numbers

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• disease in Texas caused ~40% decrease in production from 296 tons similarity (9). Three functional domains are responsible for the
• (7). The mature B-cell features a large central vacuole, which recognition receptor (PRRs) and hemocyanin, and a broad range
• Mock (n = 30) and infected (n = 40) treatments were randomly into the GEM-X microfluidics chip, along with the barcoded gel
• reverse gavage with 100 mL tryptic soy broth containing 2% sodium X Instrument to generate gel beads-in-emulsion (GEMs) and
• A pilot study was conducted to select a suitable bacteria 2.5 Alignment and processing
• CFU/shrimp was chosen for subsequent challenge. Daily, 20% of (Accession ASM4276789v1). The genome was converted to a Cell
• barcode matrices using ‘cellranger count’ with intronic counts 2.7 Pathway analysis
• UMI and < 45% mitochondrial gene expression were removed. The the compareCluster function, using cluster as the group.
• differential gene expression analysis log2FC > 0.5, and expression in at least 25% of cells. The genes were
• integration, cluster analysis was carried out using 1:20 dimensions Table 1). The libraries generated transcriptomes for 11,006 quality
• for false-discovery rate (FDR), and the list was filtered for genes expressed genes (Supplementary Table 2), and significant GO and
• with an adjusted P-value < 0.05 and a log2 fold-change (log2FC) > KEGG pathways (Supplementary Tables 3, 4) were identified.
• expressed in at least 50% of the cells in each cluster. subunit (LOC113830073) and hemocyanin subunit-like
• infected shrimp and two mock treated shrimp (A). The total mortality for the infected group was 30.0% compared to 14.3% in the mock treated
• Single-cell atlas of L. vannamei hepatopancreatic cells (n = 3). (A) The nine clusters are presented with a tSNE plot. (B) Dot plot of the top five
• (LOC113823617) (Figure 2A, Supplementary Table 2). The DE factor (ALF; LOC113800363, LOC113820510, LOC113810108,
• energy production. Hep2, despite being a distinct cluster, only has receptor signaling pathway (pvm04620) (Supplementary Table 4).
• Supplementary Table 2). The cells express hemocyanin vacuole (GO:0005773) (Supplementary Table 3), and KEGG
• (GO:0010876, GO:0006869) (Supplementary Table 3). suggest that these cells internalize substances from the external
• DNA-directed RNA polymerases I, II, and III subunit Rpb8 (Figure 2A, Supplementary Table 2). Furthermore, this cluster highly
• Supplementary Table 2). Enriched GO pathways include member A (LOC113812977; Clec7a), alpha-N-acetylgalactosamine-
• membrane (GO:0030659) (Supplementary Table 3) while the galactose-specific lectin nattectin-like (Nattectin-like; LOC138864390).
• (pvm00531) and lysosome (pvm04142) (Supplementary Table 4). (GO:0009057) and endopeptidase activity (GO:0004175)
• Cells in Hep5 expressed immune related genes, such as (Supplementary Table 3) which emphasizes the cells function
• PDZ-motif protein 52 (Zasp52), muscle-specific protein 300 kDa reads/cell (Supplementary Table 1). These five libraries generated
• chain 10 (zip) (Supplementary Table 2). The most significant KEGG samples taking batch effects into consideration, for a total of 27,374
• (Supplementary Table 4). the VP AHPND infection study (Figure 3). Overall, the same
• These include uncharacterized protein (LOC138865145), gamma- datasets (Supplementary Figure 6, Supplementary Tables 5–7).
• regulator of G-protein signaling 7-like (LOC113828644) (25) and between infected shrimp (n = 3) and control shrimp (n = 5)
• Supplementary Table 2). The protein transcribed by proportions show that the infected shrimp had higher proportions
• domain of GABAb receptors”, therefore, this gene may transcribe (Control = 6.4%; Infected = 20.3%), lower proportions of HepVp3
• another GABA receptor subunit. GABA receptors are present in (Control = 16.4%; Infected = 8.3%), HepVp5 (Control = 11.1%;
• pre- and post-synaptic sites in the mammalian central nervous Infected = 5.1%), HepVp6 (Control = 8.1%; Infected = 0.6%) and
• system. Interestingly, the cells also expressed RAS oncogene family HepVp7 (Control = 7.4%; Infected = 0.7%) (Figures 4), and
• member Rab3 (rab3b), which are highly expressed by human approximately equal proportions of HepVp2 (Control = 18.1%;
• enteroendocrine cells, pancreatic endocrine cells and inhibitory Infected = 15.7%), HepVp8 (Control = 3.8%; Infected = 3.1%) and
• neurons (26, 27). Genes in the rab3 family were shown to HepVp9 (Control = 1.4%; Infected = 1.7%). The differentially
• junction protein which enables communication between adjacent analysis of GO (Figure 6, Supplementary Table 10) and KEGG
• cells, is also highly expressed. pathways (Figure 7, Supplementary Table 11).
• transcriptomic analysis (Figure 1A). The total mortality for the (Supplementary Tables 12, 13) is significant is also presented as
• infected group was 30.0% compared to 14.3% in the mock treated supplementary data.
• were positive for V. parahaemolyticus, while the control shrimp Tables 8, 9). Hemocyanin (LOC113830073; HepVp1-8) was DE for

Methods (brief)

• digestion, immunity, molting and reproduction. The mechanism by which the
• (Litopenaeus vannamei) were sampled to produce a single-cell transcriptomic
• this journal is cited, in accordance with two mocked treated shrimp were sampled for an infection study. Primary cell
• hepatopancreas (13–17). Interestingly, there is some disagreement 2.3 Sample preparation
• construction. The samples were diluted to the target concentration
• Hepatopancreatic samples were collected one hour post Dynabeads MyOne SILANE (ThermoFisher Scientific, Waltham,
• 2.2 V. parahaemolyticus origin and culture PCR. Sample cleanup and size selection of cDNA amplicons were
• The D4 VPAHPND strain, originating from Mexico, was used for sample quality and quantity were assessed using an Agilent
• 175 rpm in TSBS. The 24 hr bacterial culture was then used to with the Qubit 1X dsDNA HS Assay Kit. cDNA samples were
• the deceased shrimp hepatopancreas were sampled on the Ranger compatible format using ‘cellranger mkref’ (Cell Ranger
• cells in the same cluster were compared using the ‘FindMarkers’
• The atlas samples were integrated using Seurat. First, the most were visualized using UpSetR v1.4.0 (20).
• 0.2. Integration for the infection study were integrated using The hepatopancreas from three healthy shrimp were collected to
• harmony (v1.2.3) to account for batch effects, where atlas samples produce a single-cell transcriptomic atlas (Figure 1A). The libraries
• were in batch 1 and mock/infection samples were in batch 2. After were sequenced to a depth of ~ 41,961 reads/cell (Supplementary
• The atlas was generated from the hepatopancreas from three shrimp, whereas for the infection study, the hepatopancreas was collected from three
• prophenoloxidase (proPO; LOC138859383), anti-lipopolysaccharide in digestion.
• chain 10 (zip) (Supplementary Table 2). The most significant KEGG samples taking batch effects into consideration, for a total of 27,374

Implications

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Wiki pages this source may touch

• Fish — marine, non-predatory (sardines, anchovies, salmon, cod)
• Shellfish (shrimp, crab, lobster, clams, oysters, mussels)
• Chromium

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Update history

The five most recent substantive edits to this page, classified major (evidence or structure moved), correction (a published value or statement was wrong and has been fixed), or minor (narrative rewritten without changing the underlying evidence). Each description is derived from what the edit did to this page; the linked commit is the authoritative record, routine regeneration passes are excluded, and the full version history lives in git. When DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.