The effect of temperature on bacteria-host interactions in the freshwater crayfish, Pacifastacus leniusculus

doi:10.1016/j.jip.2018.08.001

Journal of Invertebrate Pathology

Volume 157, September 2018, Pages 67-73

https://doi.org/10.1016/j.jip.2018.08.001 Get rights and content

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Mortality was lower for crayfish infected with bacteria at 6 °C than at 22 °C.
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Phagocytosis of bacteria was higher for infected crayfish held at 6 °C than at 22 °C.
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Melanization and degranulation were faster at 22 °C than at 6 °C.

Abstract

Water temperature is known to affect many aspects of aquatic life including immune responses and susceptibility to diseases. In this context, we studied the effect of temperature on the defense system of the freshwater crayfish Pacifastacus leniusculus. Animals were challenged with two pathogenic Gram-negative bacteria, Aeromonas hydrophila and Pseudomonas gessardii, as well as the bacterial cell wall component lipopolysaccharide (LPS) at two different temperatures, cold (6 °C) and room temperature (22 °C). The immune responses were compared by means of differences in mortality, phagocytosis, bacterial clearance, and the melanization reaction of the hemolymph at these two temperatures. We observed that crayfish survival was higher at cold temperature. The mortality rate was zero at 6 °C following A. hydrophila or LPS injections. Furthermore, the bacteria were completely cleared from crayfish after they had been held at 6 °C for more than 9 days. We also observed a strong melanization reaction of hemolymph at 22 °C when stimulated with LPS, as well as with bacteria. Taken together, our results suggest that the cellular immunity is more effective at low temperature in this cold-adapted animal and pathogens are efficiently removed from the body by mean of phagocytosis.

Graphical abstract

Introduction

Invertebrates such as insects and crustaceans do not have pathogen specific adaptive immunity but they can rapidly and efficiently clear foreign intruders from the body using cellular and humoral immunity. Cellular immunity is executed by means of phagocytosis, opsonisation and encapsulation of the pathogens (Johansson and Söderhäll, 1989), whereas humoral immunity is characterized by the activation of a proPO cascade followed by the production and secretion of immune components such as antimicrobial peptides. The proPO-system is activated upon recognition of microbial cell-wall components of the pathogen, the so-called pathogen-associated molecular pattern (PAMP), for example lipopolysaccharides (LPS) from Gram-negative bacteria, peptidoglycans (PGN) from Gram positive bacteria or β-1,3-glucans from fungi (Cerenius et al., 2008, Cerenius and Söderhäll, 2018, Söderhäll and Cerenius, 1998). The semigranular cells (SGCs) and granular cells (GCs) release granules packed with the components of PO activation cascade by exocytosis in contact with PAMPs (Sricharoen et al., 2005) into the hemolymph, where the system becomes activated and initiates the melanization reaction. The final step of the melanization reaction is the conversion of inactive zymogen proPO to active phenoloxidase (PO) by the prophenoloxidase activating enzyme (ppA) (Wang et al., 2001), resulting in the formation of melanin pigments as well as toxic products such as quinones and other derivatives of DOPA or dopamine (Cerenius et al., 2008, Söderhäll and Cerenius, 1998). Due to the production of toxins, this system is tightly regulated at different steps along the cascade by various inhibitors (Cerenius et al., 2008, Liang et al., 1997, Noothuan et al., 2017, Söderhäll et al., 2009).

In this study, we evaluated the effect of temperature on the immune response of the freshwater crayfish Pacifastacus leniusculus. In order to stimulate the immune system of P. leniusculus, we injected the bacterial strains Aeromonas hydrophila B1 or Pseudomonas gessardii, which differ in virulence. Both species are Gram-negative, rod-shaped pathogenic bacteria, naturally occurring in aquatic environments. In addition to these bacteria, we tested the Gram-negative bacterial cell wall component LPS to induce immunity. We compared the death rates, bacterial clearance, phagocytosis and melanization between two groups of animals that were maintained at low and high temperatures.

Section snippets

Animals

Freshwater crayfish (P. leniusculus) from Lake Erken in Sweden, were maintained in tanks with aerated running tap water at 10 °C. Only intermolt and apparently healthy crayfish were used in the experiments. Before the experiments, the animals were transferred to smaller 6-liter tanks with aerated water at 6 °C or 22 °C, at a maximum of four crayfish per container. Animals were maintained at these temperatures for at least 3 days prior to the experiments to adjust to their new temperatures.

Mortality data

We first tested whether temperature had an effect on the mortality of crayfish after infecting the crayfish with the virulent bacterial strains A. hydrophila B1, and P. gessardii (Jiravanichpaisal et al., 2009) or with the bacterial cell wall component LPS. For this purpose, two groups of crayfish were maintained at two different temperatures: 6 °C and 22 °C. After injection with A. hydrophila B1, mortality was zero in the animals held at 6 °C for over a week, whereas animals held at 22 °C died

Discussion

The freshwater crayfish, P. leniusculus, occurs naturally in lakes with low temperatures, rarely higher than 20 °C (Bohman et al., 2016). The effect of temperature on white spot syndrome virus (WSSV) infection has been previously described in P. leniusculus (Jiravanichpaisal et al., 2004). Crayfish injected with this highly virulent virus survived at temperatures lower than 12 °C but mortality was high at room temperature. In order to determine if immune response to bacterial infections show

Conclusion

Our study reveals that the immune system of crayfish, regulation of immune responses and ability of the host to combat invaders, works most efficiently at 6 °C, the approximate ambient temperature of the aquatic crayfish habitat. Increasing lake water temperatures may therefore increase the risk that bacterial infections will have significantly more serious consequences.

Acknowledgements

This work was supported by grants from Swedish Science Research Council to KS (621-2012-2418) and Formas (2011-606).

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

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The effect of temperature on bacteria-host interactions in the freshwater crayfish, Pacifastacus leniusculus

Highlight

Abstract

Graphical abstract

Introduction

Section snippets

Animals

Mortality data

Discussion

Conclusion

Acknowledgements

Conflict of interest

Trends Immunol.

Dev. Comp. Immunol.

J. Invertebr. Pathol.

Fish Shellfish Immunol.

Biochem. Biophys. Res. Commun.

Parasitol. Today Pers. Ed.

Biochim. Biophys. Acta

Dev. Comp. Immunol.

Dev. Comp. Immunol.

J. Biol. Chem.