Epidemiology of Noble Pen Shell ( Pinna nobilis L. 1758) Mass Mortality Events in Adriatic Sea Is Characterised with Rapid Spreading and Acute Disease Progression

Abstract

From May to October 2019, multiple mass mortality events (MMEs) of Pinna nobilis were observed along Croatian coastline starting from the south-east and rapidly progressing in north-western direction. Time dynamics of the MMEs closely followed general speed and direction patterns of surface sea-currents, advancing approximately 350 km in less than 3 months. Surveillance, clinical evaluation, and sample collection were performed on multiple sites with various degrees of mortality rates. Moribund P. nobilis individuals were collected and subjected to pathological, molecular, and microscopical investigation. Affected animals were positive for Mycobacterium in 70% of the individuals, and Haplosporidium pinnae was present in 58% of the cases. Observed pathological lesions were most severe where concurrent presence of both pathogens was confirmed (in 45.8% of moribund individuals). Moderate to strong lesions were observed in animals positive for Mycobacterium only (25% of cases), and lesions were absent or minor to moderate when only H. pinnae was confirmed (16% of cases). Considering the rapid and severe spread of the MMEs, the areas less exposed to major sea currents appeared to be at lower risk of pathogen transmission. Surveillance activities along the Croatian coastline identified several P. nobilis populations in such "lower risk" areas without apparent mortality or clinical symptoms. Such areas are of particular interest as source of potentially healthy individuals to support active recovery actions.

Keywords: Haplosporidium pinnae; MMEs; Mycobacterium; epidemiology.

Figure 1

Surveillance sites of Pinna nobilis mortality in Croatia from May 2019 to January 2020 (a) and details sites at Telašćica Bay (b) with the monitoring site 1: Kobiljak Bay; monitoring site 2: Kršovica Bay and monitoring site 3: Magrovica Bay.

Figure 2

Timeline map of the spread of the MMEs detected from May 2019 to January 2020 in areas along the Croatian coastline and during related sampling periods.

Figure 3

Gross examination of healthy (A) and diseased animals (B,C) from different areas. (A) healthy individual showing visible colourful developing gonad (G), gills (Gi), and deep brown digestive tissue (DT); (B) diseases specimen from Telašćica Bay shows an evident cyst with liquid content located on the left side of the digestive tissue (DT, arrowheads); (C) empty watery tissue in a small individual from Mali Ston Bay. G: gonad; AM: Adductor Muscle; M: Mantle; By: Byssus; K: Kidney; Gi: Gill; G: Gonad; Po: Pallial organ; F: Foot; LP: Labial Palps.

Figure 4

Smear from digestive gland cyst of the individual from Telašćica Bay: different developing plurinucleate pre-sporulation phases of H. pinnae parasite. Note multinucleate plasmodium where some cytoplasmic compartmentalisation is distinguished (arrowheads).

Figure 5

Histopathology of specimens of pen shell P. nobilis in August 2019 in Sakarun and Telašćica Bay. (A) Co-representation of plasmodial phases of H. pinnae (arrows) and Mycobacterium (arrowheads) in Sakarun (BC: Brown cells); (B) diffuse presence of initial phases of H. pinnae (arrows) in the digestive gland (DT: digestive tubules); (C) inflammatory nodules containing Mycobacterium (arrowheads) in Sakarun; (D) colliquative necrosis of the digestive tubules; (E,F) Presence of Mycobacterium sp. with a grading of infection of score 2 in Telašćica Bay within inflammatory nodules using routine ZN (E) and MZN stain (F).

Figure 6

Histopathology pen shell P. nobilis in October and November 2019. (A,B) Presence of ciliates (arrowheads) in the gill (G) accompanied by PAS-BA positive secretions (B) and granulocytes (gr); (C) Advanced phases of development of H. pinnae (Masson Trichrome); (D) unknown helminth egg in the connective tissue surrounding the gonad (Ov: ovocytes) in Kaštela in November 2019.

Figure 7

Neighbor-Joining tree of the rRNA sequences of Haplosporidium sp. The bootstrap values (1000 replicates) are shown next to the branches. All ambiguous positions were removed for each sequence pair (pairwise deletion option). The accession numbers of the sequences used are reported in the tree. Minchinia sp. was used as outgroup.

Figure 8

Neighbor-Joining tree of the 16S rRNA sequences of Mycobacterium of P. nobilis. The bootstrap values (1000 replicates) are shown next to the branches. All ambiguous positions were removed for each sequence pair (pairwise deletion option). The accession numbers of the sequences used are reported into the tree. Nocardia crassostreae was used as outgroup.