Abstract
Early changes in hemocyte proteins in freshwater crayfish Pacifastacus leniusculus, in response to an injection with the fungal pattern recognition protein β-1,3-glucan (laminarin) were investigated, as well as changes after saline (vehicle) injection and in naïve animals. Injection of saline resulted in rapid recruitment of granular hemocytes from surrounding tissues, whereas laminarin injection on the other hand induced an initial dramatic drop of hemocytes. At six hours after injection, the hemocyte populations therefore were of different composition. The results show that mature granular hemocytes increase in number after saline injection as indicated by the high abundance of proteins present in granular cell vesicles, such as a vitelline membrane outer layer protein 1 homolog, mannose-binding lectin, masquerade, crustin 1 and serine protease homolog 1. After injection with the β-1,3-glucan, only three proteins were enhanced in expression, in comparison with saline-injected animals and uninjected controls. All of them may be associated with immune responses, such as a new and previously undescribed Kazal proteinase inhibitor. One interesting observation was that the clotting protein was increased dramatically in most of the animals injected with laminarin. The number of significantly affected proteins was very few after a laminarin injection when compared to uninjected and saline-injected crayfish. This finding may demonstrate some problematic issues with gene and protein expression studies from other crustaceans receiving injections with pathogens or pattern recognition proteins. If no uninjected controls are included and no information about hemocyte count (total or differential) is given, expressions data for proteins or mRNAs are very difficult to properly interpret.
Highlights
Crayfish, as with other invertebrates, lack an adaptive immune system and are reliant on their blood cells in response to pathogens or injury
The global proteome in hemocytes of healthy male P. leniusculus crayfish was investigated at six hours after injection of saline or laminarin, a β-1,3-glucan derived from the cell wall of the brown algae Laminaria digitata
Proteome mapping of the total hemocyte count through NanoLC-mass spectroscopy (MS)/MS was performed, and the proteomes of five saline, five laminarin-injected and five uninjected individuals were compared. This approach was chosen as a means to characterize an early hemocyte response to β-1, 3-glucan, as well as to show the effects achieved by injection of saline alone since this treatment is widely used as a control for injection experiments in crustaceans
Summary
As with other invertebrates, lack an adaptive immune system and are reliant on their blood cells in response to pathogens or injury. The crayfish blood cells, called hemocytes, are the main effectors of the immune responses. After an infection or injury, the number of circulating hemocytes is rapidly depleted [20,21] This leads to a need for new hemocytes to be released from the hematopoietic tissue (HPT), located as a sheet over the dorsal part of the stomach [22,23]. It is of high importance to understand how the hemocyte composition in the circulation is affected by a challenge with pathogens or pathogen-associated molecules, as well as any control injection as the responses may be very different. This issue is further addressed in the present study
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