Impact of acute stress on antimicrobial polypeptides mRNA copy number in several tissues of marine sea bass (Dicentrarchus labrax)

Abstract

Background: In comparison to higher vertebrates, fish are thought to rely heavily on innate immune system for initial protection against pathogen invasion because their acquired immune system displays a considerably poor immunological memory, and short-lived secondary response. The endogenous antimicrobial polypeptides (AMPPs) directly and rapidly killing pathogens such as bacteria, fungi, parasites, and viruses are included within the realm of innate defenses. In addition to piscidins, AMPPs that in recent years have been shown to be commonly linked to innate defense, are histones and their polypeptide fragments, and peptides derived from the respiratory protein hemoglobin. There is evidence that a number of stresses lead to significant regulation of AMPPs and thus their monitoring could be a highly sensitive measure of health status and risk of an infectious disease outbreak, which is a major impediment to the continued success of virtually all aquaculture enterprises and is often the most significant cause of economic losses.

Results: We firstly isolated and deposited in Genbank database the cDNA sequences encoding for hemoglobin-β-like protein (Hb-LP) [GeneBank: JN410659], H2B histone-like protein 1 (HLP1) GenBank: JN410660], and HLP2 [GenBank: JN410661]. The "de novo" prediction of the three-dimensional structures for each protein is presented. Phylogenetic trees were constructed on Hb-LP, HLP1, and HLP2 sequences of sea bass and those of other teleost, avian, reptiles, amphibian and mammalian species. We then used real time RT-PCR technology to monitor for the first time in sea bass, dynamic changes in mRNA copy number of Hb-LP, HLP1, HLP2, and dicentracin in gills, skin, eyes, stomach and proximal intestine in response to acute crowding/confinement stress. We showed that acute crowding stress induces an increase in the expression levels of the aforementioned genes, in gills and skin of sea bass, but not in other tissues, and that this expression patterns are not always rapidly reversed upon re-exposure to normal conditions.

Conclusion: The higher expression of the four target genes in gills and skin of sea bass suggests that this AMPP represents a first and immediate line of defense in combating pathogens and stressors since these tissues constitute the first physiological barriers of the animal.

Figure 1

Dicentracin gene expression levels in tissues of D. labrax. The dicentracin mRNA copy number was measured by real-time PCR in gills, skin, eyes, stomach and gut, and it was normalized as a ratio to 100 ng total RNA. Fish were sampled before stress (control), after 4 hours of confinement/crowding (stress), and then after 24 hours of recovery from stress. The means of five animals in each group are shown. Bars indicate standard error of the mean. Differences were determined by one-way ANOVA. Different letters indicate significantly different means (p < 0.05) between control, stressed and recovered fish groups.

Figure 2

Hb-LP gene expression levels in tissues of D. labrax. The Hb-LP mRNA copy number was measured by real-time PCR in gills, skin, eyes, stomach and gut, and it was normalized as a ratio to 100 ng total RNA. Fish were sampled before stress (control), after 4 hours of confinement/crowding (stress), and then after 24 hours of recovery from stress. The means of five animals in each group are shown. Bars indicate standard error of the mean. Differences were determined by one-way ANOVA. Different letters indicate significantly different means (p < 0.05) between control, stressed and recovered fish groups.

Figure 3

HLP1 and HLP2 gene expression levels in tissues of D. labrax. The HLP1 and HLP2 mRNA copy number was measured by real-time PCR in gills, skin, eyes, stomach and gut, and it was normalized as a ratio to 100 ng total RNA. Fish were sampled before stress (control), after 4 hours of confinement/crowding (stress), and then after 24 hours of recovery from stress. The means of five animals in each group are shown. Bars indicate standard error of the mean. Differences were determined by one-way ANOVA. Different letters indicate significantly different means (p < 0.05) between control, stressed and recovered fish groups.

Figure 4

DNA binding sites of the "de novo" predicted tertiary structure of D. labrax AMPPs. (HLP1 is A, HLP2 is B and HB-like protein is C). DNA binding sites are presented in light blue with the surface.

Figure 5

The functional sites of the "de novo" predicted tertiary structure of D. labrax AMPPs. (HLP1 is A, HLP2 is B and HB-like protein is C). Colour code: residues predicted to be phosphorylated: pink = Y, magenta = S, purple = T; epsilon-glycosylated = green; N-glycosylated = light blue (only in the Hb-LP); bound to DNA = blue (only in HLP1 and HLP2). The Thr residues shown in orange in HLP1 can be phosphorylated or O-glycosylated.

Figure 6

Phylogenetic tree comparing the sequence of Hb-LP in sea bass (Dicentrarchus labrax) with those of other vertebrate species. The scale bar refers to evolutionary distances in substitutions per site. The numbers at tree nodes refer to percentage bootstrap values after 1000 replicates. GenBank accession numbers for Hb-LP cDNA sequence comparisons are: U60902.1 [thick-tailed bushbaby (Otolemus crassicaudatus)]; NG000007.3 [human (Homo sapiens)]; JO3642.1 [North American opossum (Didelphis virginiana)]; X64179.1 [golden hamster (Mesocricetus auratus)]; U63145.1 [red-footed tortoise (Geochelone carbonaria); M73995.1 [red junglefowl (Gallus gallus)]; GQ272085.1 [speckled teal (Anas flavirostris flavirostris); X03142.1 [african clawed frog (Xenopus laevis)]; EU877979.1 [marine toad (Bufo marinus); JN410659 [sea bass (Dicentrarchus labrax)]; AB043642.1 [Japanese amberjack (Seriola quinqueradiata)]; AY190698.1 [red seabream (Pagrus major)]; AJ277207.1 [seabream (Sparus aurata)]; DQ109665.1 [red drum (Sciaenops ocellatus)].

Figure 7

UPGMA phylogenetic tree depicting the evolutionary relationship of vertebrate HLP1 proteins. The tree was constructed using the neighbour-joining method based on the alignment of the complete cDNA coding sequences of known vertebrate Hb-LPs. The scale bar refers to evolutionary distances in substitutions per site. Bootstrap values (1000 replicates) indicating the occurrence of nodes are reported above each branch in the figure. GenBank accession numbers for HLP1 cDNA sequence comparisons are: XM844635.2 [Dog H2B (Canis lupus familiaris)]; XM002930226.1 [Giant panda (Ailuropoda melanoleuca)]; XM_001505029.2 [Horse (Equus caballus)]; XM_001367484.1 [Gray short-tailed opossum (Monodelphis domestica)]; XM_001515033.1 [Platypus (Ornithorhynchus anatinus); XM_003259153.1 [Northern white-cheeked gibbon (Nomascus leucogenys)]; NM_080593.2 [Human (Homo sapiens)]; XM_003311114.1 [Chimpanzee (Pan troglodytes); XM_003416418.1 [African savanna elephant (Loxodonta africana)]; AB124798.1 [Japanese gliding frog (Rhacophorus schlegelii)]; XM_003226650.1 [Green anole (Anolis carolinensis)]; XM_427116.1 [Red jungle fowl (Gallus gallus)]; XM_003201926.1 [Turkey (Meleagris gallopavo)]; JN410660 [Sea bass (Dicentrarchus labrax)]; GU982558.1 [orange-spotted grouper (Epinephelus coioides)]; X02916.1 [Rainbow trout (Onchorhynchus mykiss)]; AB435638.1 [Japanese medaka (Oryzias latipes)]; XM_002666939.1 [Zebrafish (Danio rerio)].

Figure 8

Phylogenetic tree comparing the sequence of HLP2 in sea bass (Dicentrarchus labrax) with those of other vertebrate species. The scale bar refers to evolutionary distances in substitutions per site. The numbers at tree nodes refer to percentage bootstrap values after 1000 replicates. GenBank accession numbers for HLP2 cDNA sequence comparisons are: XM_002194486.1 [zebra finch (Taeniopygia guttata)]; M17018.1 [red jungle fowl (Gallus gallus)]; X06128.1 [duck (Anas platyrhynchos)]; NM_001016946.2 [western clawed frog (Xenopus (Silurana) tropicalis)]; AY158905.1 [mouse (Mus musculus)]; XM_002721618.1 [rabbit (Oryctolagus cuniculus)]; XM_001364664.1 [gray short-tailed opossum (Monodelphis domestica)]; XM_545380.1 [dog (Canis lupus familiaris)]; XM_527252.2 [chimpanzee (Pan troglodytes); NM_005325.3 [human (Homo sapiens)]; JN410661 [sea bass (Dicentrarchus labrax)]; AY184811.1 [goldfish (Carassius auratus)]; XM_693405.3 [zebrafish (Danio rerio)]; X02624 [rainbow trout (Salmo gairdneri) (Onchorhynchus mykiss)].