The Immune Response of the Invasive Golden Apple Snail to a Nematode-Based Molluscicide Involves Different Organs

Abstract

The spreading of alien and invasive species poses new challenges for the ecosystem services, the sustainable production of food, and human well-being. Unveiling and targeting the immune system of invasive species can prove helpful for basic and applied research. Here, we present evidence that a nematode (Phasmarhabditis hermaphrodita)-based molluscicide exerts dose-dependent lethal effects on the golden apple snail, Pomacea canaliculata. When used at 1.7 g/L, this biopesticide kills about 30% of snails within one week and promotes a change in the expression of Pc-bpi, an orthologue of mammalian bactericidal/permeability increasing protein (BPI). Changes in Pc-bpi expression, as monitored by quantitative PCR (qPCR), occurred in two immune-related organs, namely the anterior kidney and the gills, after exposure at 18 and 25 °C, respectively. Histological analyses revealed the presence of the nematode in the snail anterior kidney and the gills at both 18 and 25 °C. The mantle and the central nervous system had a stable Pc-bpi expression and seemed not affected by the nematodes. Fluorescence in situ hybridization (FISH) experiments demonstrated the expression of Pc-bpi in circulating hemocytes, nurturing the possibility that increased Pc-bpi expression in the anterior kidney and gills may be due to the hemocytes patrolling the organs. While suggesting that P. hermaphrodita-based biopesticides enable the sustainable control of P. canaliculata spread, our experiments also unveiled an organ-specific and temperature-dependent response in the snails exposed to the nematodes. Overall, our data indicate that, after exposure to a pathogen, the snail P. canaliculata can mount a complex, multi-organ innate immune response.

Keywords: Phasmarhabditis hermaphrodita; Pomacea canaliculata; eco-immunology; hemocytes; immunity; invertebrate; molluscicide; mollusk; pest control.

Figure 1

Concentration and temperature-dependent effects of the Nemaslug^® molluscicide. The bar chart reports the mortality of apple snail exposed to the Phasmarhabditis hermaphrodita worm, that increases with Nemaslug^® concentration. At 18 (dark gray) and 25 (black) °C, lethal effects on the apple snails were observed, whereas at 30 °C (light gray, in the insert) incubation temperature the molluscicide was ineffective. *** = p < 0.001. Fisher’s exact test. In the case of a lack of nematode effects, no mortality was expected.

Figure 2

Histological sections of P. canaliculata anterior kidney collected from control (A,B) or treated (C,D) snails and stained by Masson’s trichrome staining. (A,B) In the anterior kidney, parenchyma is organized in ramified protrusions with a connective core and an external layer of epithelial cells. (C,D) A nematode is visible inside the renal parenchyma (green asterisks). No evident signs of encapsulation or hemocyte accumulation are visible.

Figure 3

Histological sections of P. canaliculata gills (also known as ctenidia) collected from control (A,B) or treated (C,D) snails and stained with Masson’s trichrome staining. (A,B) Gills present parallel lamellae with a loose connective core (blue) and an external ciliated epithelium (red). (C,D) A nematode is visible between lamellae (green asterisks). (D) The gill epithelium remained intact and also at higher magnification the tissue organization and the cilia did not seem affected by the presence of the nematode.

Figure 4

Histological sections of P. canaliculata (A,B) mantle and (C,D) pedal ganglia stained with Masson’s trichrome staining. (A). The mantle skirt tissue in control snails is mainly constituted by connective fibers, colored in blue, more densely packed in proximity of the external epithelium. (B) In treated snails, the mantle organization appeared well-conserved with no infiltration of nematodes. A portion of muscular tissue is visible in red. (C,D) A representative example of pedal ganglion showing highly dense neural tissue with neuron cell bodies and axons (red) clearly distinguishable from the peripheral connective tissue (blue). (D) Ganglia collected from treated snails display unaltered organization, and no P. hermaphrodita were observed either inside the neural tissue or close to the ganglion surface.

Figure 5

(A). Multiple sequence alignment of vertebrate and invertebrate BPI proteins using MUSCLE software. Homo sapiens (NP_001716.2), Pogona vitticeps (XP_020659320.1), Xenopus tropicalis (NP_001015694.1), Gadus morhua (AAM52336.1), P. canaliculata (XP_025114931.1), Crassostrea gigas (AAN84552.1), Eisenia andrei (AFI44048.1), and Amphimedon queenslandica (XP_019854892.1). Conserved amino acids are highlighted in yellow (identical amino acids with 70% consensus) and in green (similar amino acids with 70% consensus). The two conserved cysteine residues, putatively forming the disulfide bond, are boxed with red lines. The LPS-binding domain is boxed with a solid black line, and the proline-rich domain, connecting the C- and N-terminal domains, is boxed with a dashed black line. The blue box indicates the first and the last amino acids corresponding to the mRNA sequence targeted by the probe for in situ hybridization (B). Sequence of primers used for probe synthesis and essential information about the probe for in situ hybridization.

Figure 6

qPCR analysis of Pc-bpi expression performed on anterior kidney, gills, mantle, and ganglia of P. canaliculata at 18, 25, and 30 °C. The changes in mRNA expression between control and treated animals were calculated with the 2^-ΔΔCt method. p-value is reported only in the case of significant difference between control and treated samples. Each dot represents a single animal, bars represent mean +/- standard deviation.

Figure 7

Pc-bpi constitutive expression in circulating hemocytes after cytocentrifugation and fluorescence in situ hybridization (FISH) experiments. (A) Pc-bpi-positive (green) hemocytes are evident. (B) Detail of positive hemocytes. (C) Negative control. Bars: (A) 5 µm,(B) 3 µm, (C) 4 µm.