1. Introduction

The increasing interconnectedness of the modern world brings unintended consequences to natural ecosystems. Economic ties between countries facilitate the intercontinental transport of goods across the world’s oceans, with commercial shipping vessels playing a crucial role in connecting ecosystems that previously had no natural link. These vessels can inadvertently transport non-native organisms on their hulls, in ballast water, or alongside cargo [1–3]. Species that survive transport may successfully establish new populations, and a few will subsequently spread beyond their entry ports and potentially become invasive (e.g., sea walnut [4]). Another common pathway for the introduction of non-native marine species is through aquaculture. Most of the species farmed are non-native and escapes often occur in mariculture because of poor facility maintenance, intense storms (e.g., hurricanes), and damage to nets or cages by other animals (e.g., Atlantic salmon and rainbow trout [5]). The import of farmed bivalves has also resulted in the unintentional introduction of several non-native species, such as the American whelk tingle that was introduced to England with the American oyster [6] and the introduction of the slipper limpet in Europe associated with the Pacific oyster [7]. Once a marine non-native species becomes established, its eradication is complex, and even if successful, the species is likely to be reintroduced [8]. Thus, most efforts to date focus on preventing species introductions and mitigating the adverse effects of non-native species on natural ecosystems and ecosystem services. For the aquaculture industry, mitigation includes biofouling control of native and non-native species and was estimated to represent 5%–30% of production costs [9–11].

Ascidians, or sea squirts, are filter-feeding animals found in benthic communities around the globe. These animals are dispersed long distances attached to ship hulls, buoys, and nets [12] and are prevalent and abundant in a variety of artificial substrates, including harbors and aquaculture facilities [9, 13–18], where they directly compete with farmed animals for food [19] and substrate [20], causing significant economic losses [21, 22]. Non-native ascidians are characterized by rapid growth rates and long reproductive seasons [23–26], releasing larvae into the water column that settle within a few days [27, 28]. Thus, ascidians cannot be eradicated once present on a shellfish farm and mitigation techniques must be applied to reduce their impact [29, 30].

The most common method of combating ascidian fouling on oyster cages is by flipping the cages periodically (every 2–3 weeks), exposing ascidians on one side of the cage to the open air to cause desiccation, ultimately leading to the death of the animals [31, 32]. Other regularly used techniques include power washing [33], manual removal by scraping biofouling organisms off lines and cages [34], and removing the whole cage from the water to dry completely after shellfish have been harvested [35]. Other methods, such as chemical treatments with lime, bleach, or acetic acid, are less frequently utilized due to their potential adverse effects on the farmed animals [34, 36–39]. The introduction of natural predators (biological treatments) has also been attempted to mitigate biofouling with mixed results [40, 41].

The shellfish aquaculture industry is a small but important industry in North Carolina’s (NC) coastal communities, contributing over $31 million to the state’s economy in 2022 [42]. Currently, most producers grow oysters, with a few also growing clams and scallops, in the water column on a free-flow system where oyster seeds are placed into floating cages or cages suspended from the bottom. The state allowed the use of public waters to grow shellfish commercially in 1858 [43]. Since then, the industry has grown rapidly, with projected contributions of $100 million to the economy by 2030 [42]. Although the negative impacts of ascidians in aquaculture facilities are well known, no study to date has characterized ascidian assemblages in NC shellfish farms. Here, we visited shellfish farms along the NC coast and at each site (1) identified all ascidians encountered using key morphological characters, (2) determined the introduced status of each species, and (3) calculated the abundance of each species at each farm. Equivalent data was also collected from marinas to compare ascidian composition and abundance between the two habitats.

2. Material and Methods

2.1. Study Sites

Five aquaculture sites were surveyed (Figure 1 and Table 1), each composed of one to four mooring lines: (1) Morris Family Shellfish Farm, located on Nelson Bay, and growing oysters exclusively. (2) Cedar Island Oyster Company, located next to Cedar Island Bay and separated from the Atlantic Ocean by Cape Lookout National Seashore produce oysters. (3) Waterman’s Choice Topsail Sound Farm is shielded from the Atlantic by Topsail Beach, NC. This farm grows both clams and oysters, but only oyster cages were surveyed since clams were grown in fully submerged bags. (4) NC State Center for Marine Science and Technology (CMAST), located in Bogue Sound, behind Atlantic Beach, NC. This was the smallest deployment, with only three cages on one mooring line and exclusively growing scallops. (5) UNCW’s Shellfish Hatchery, located behind the Masonboro Island Reserve. The hatchery grows both oysters and scallops, but only the oyster cages were deployed during this study.

Table 1. Study sites, GPS coordinates, habitat type, sampling date, salinity, temperature (°C) at the sampling time, species richness (SpR), and Shannon index (H).

Study site	GPS coordinates	Habitat	Sampling date	Salinity	Temperature (°C)	SpR	H
Cedar Island Oyster Company	35° 00′ 23.926″ N 76° 18′ 30.356″ W	Shellfish farm	October 23rd, 2022	30	17.5	1	0
Morris Family Shellfish Farm	34° 52′ 17.784″ N 76° 23′ 37.391″ W	Shellfish farm	October 22nd, 2022	35	19.0	2	0.3010
Morehead City Yacht Basin	34° 43′ 16.577″ N 76° 42′ 14.367″ W	Marina	October 15th, 2022	35	21.5	4	0.2919
Anchorage Marina	34° 42′ 0.671″ N 76° 43′ 47.36″ W	Marina	October 16th, 2022	34	20.9	2	0.1470
CMAST	34° 43′ 20″ N 76° 45′ 36″ W	Shellfish farm	October 6th, 2023	30	24.7	5	0.5443
Waterman’s Choice Topsail Sound Farm	34° 22′ 50″ N 77° 37′ 31″ W	Shellfish farm	October 5th, 2023	35	24.7	6	0.5892
Harbor Village Marina	34° 23′ 18″ N 77° 38′ 13″ W	Marina	October 5th, 2023	34	25.7	4	0.4278
Seapath Marina	34° 12′ 48″ N 77° 48′ 22″ W	Marina	October 16th, 2023	36	21.0	12	0.9317
UNCW Shellfish Hatchery	34° 8′ 26″ N 77° 51′ 50″ W	Shellfish farm	October 9th, 2023	35	21.0	3	0.2645

• Abbreviation: CMAST, NC State Center for Marine Science and Technology.

In addition, four nearby marinas were surveyed (Figure 1 and Table 1): (1) Morehead City Yacht Basin, (2) Anchorage Marina, (3) Harbor Village Marina, and (4) Seapath Marina. Morehead City Yacht Basin is dedicated to sport fishing with vessels that travel along the coast for regional tournaments. The other three marinas specialize in private recreational boating. Seawater temperature and salinity were recorded at all sites at the time of sampling (Table 1). The marinas were chosen for their proximity to the surveyed farms and accessibility. No marinas exist in the vicinity of the two northernmost farms visited (Figure 1).

3. Results

Six ascidian species were found in the surveyed shellfish farms (Figure 2): three species were native to NC: Clavelina oblonga (Herdman, 1880); Perophora viridis [54]; and Molgula manhattensis (De Kay, 1843). Two species, Distaplia bermudensis (Van Name, 1902) and Styela plicata (Lesueur, 1823), were considered cryptogenic, and the last species, Didemnum sp., was only identified at the genus level because zooid contraction made characterization of some key morphological characters unreliable. The most common and abundant species in shellfish farms was the cryptogenic S. plicata, observed in all the sites surveyed (Figure 3). The native C. oblonga was also common, although it was only observed at three of the five sites visited and at lower abundances than S. plicata. The native P. viridis and M. manhattensis were observed in two shellfish farms, while D. bermudensis and Didemnum sp. were only observed at one site (Figure 3). All species observed in shellfish farms were also observed in marinas (Figure S1). However, six additional species were observed in the marinas: three native species, Eudistoma capsulatum (Van Name, 1902); Didemnum lutarium (Van Name, 1910); and Distaplia corolla (Monniot, 1974), the introduced, Polyandrocarpa anguinea (Sluiter, 1898), and the cryptogenic, Distaplia stylifera (Kowalevsky, 1874) and Botrylloides niger [55]. The Shannon index ranged from 0.9317 in the Seapath Marina, where all 12 ascidian species were observed, to 0 in Morris Family Shellfish Farms, where only S. plicata was recorded (Table 1).

Styela plicata reached numbers as high as 423 individuals per m² at Cedar Island Oyster Company and as high as 376 individuals per m² at the Morehead City Yacht Basin. The species was prevalent in all surveyed sites, with the lowest abundance recorded at the UNCW Shellfish Hatchery (29 individuals per m²). Other species common in shellfish farms and marinas were the native species P. viridis and C. oblonga. Neither species was recorded at all sites, and both were much more abundant in marinas than farms (P. viridis: 9.6 colonies/m² ± 14.5 SD in marinas and 2.5 ± 3.5 in farms; C. oblonga: 34 colonies/m² in marinas ± 23 SD and 1.8 ± 2 in farms; Figures 3 and S1). Colonial species like D. bermudensis and D. corolla were more common in marinas, while M. manhattensis was observed in two shellfish farms, but only one marina (Figures 3 and S1).

Presence–absence analysis based on the Bray–Curtis similarity matrix revealed that species composition at the two southernmost surveyed farms, UNCW Shellfish Hatchery, and Waterman’s Choice Topsail Sound Farm, were the most similar (Figure 4A). However, ascidian assemblages at these two farms were more comparable to that observed at a northern marina (Anchorage Marina) than at the closest marinas (Seapath Marina and Harbor Villa Marina; Figures 1 and 4A). The Cedar Island Oyster Company and the Morris Family Shellfish Farm in northern NC are located only 16.9 km apart, and the ascidian composition at both sites was most similar. Finally, ascidian assemblages in CMAST, the only non-oyster farm, resembled that of a nearby marina, Morehead City Yacht Basin (Figure 4A).

When considering species abundances at each surveyed site, similarity groupings differed from those based on presence–absence data. Both Morris Family Shellfish Farms and Cedar Island Oyster Company hosted communities that were more similar to those observed in marinas than to each other (Figure 4B). Ascidian communities at CMAST (in northern NC) and Waterman’s Choice Topsail Sound Farm (in southern NC) were similar to each other, while the ascidian community at UNCW Shellfish Hatchery was distinct from all other sites (Figure 4B). The UNCW Shellfish Hatchery also had the least number of ascidians per m² (Figure 3). Overall, there were no significant differences in species composition (p = 0.37) or abundance (p = 0.46) between marinas and aquaculture farms.

4. Discussion

This study is the first to characterize ascidian assemblages in shellfish farms in NC where they are considered pests. Six ascidian species were observed: two cryptogenic species, S. plicata and D. bermudensis; three native species, C. oblonga, M. manhattensis, and P. viridis; and a didemnid species. The most common species in terms of presence and abundance were the solitary ascidian S. plicata, and the colonial C. oblonga. Six additional species were observed in marinas, but overall, there were no significant differences in ascidian assemblages between the surveyed shellfish farms and the marinas.

All species observed in shellfish farms have an extensive distribution range in NC, including marinas [49, 50] and seagrass meadows [56]. The solitary S. plicata dominated all five of the shellfish farms, being the only ascidian present at the two northernmost study sites and the only species found in all nine sample sites visited. Styela plicata has also been noted as the most common species in NC marinas [49, 50] and seagrass beds [56]. In fact, S. plicata has been introduced in many regions of the world [57, 58], and although its origin was hypothesized to be in the NW Pacific Ocean [45, 57, 59, 60], recent data does not support this claim [58, 61]. The species is highly resilient to a wide range of environmental conditions, including temperature and salinity changes [62], pollution [63], and even hurricane impacts [50]. The other cryptogenic species, D. bermudensis, was first described in the Bermuda Islands, and although less conspicuous than S. plicata, the species has populations in the Mediterranean Sea, Brazil, and the southeastern United States [15, 49, 64, 65]. In NC, the species has been observed both in marinas and natural reefs [66].

Similarly, the three native species observed in shellfish farms have established populations in many regions of the world. Clavelina oblonga has been reported in Brazil, Panama, the Azores, Africa, Italy, and Spain [67–70] and is found on natural and artificial substrates [68], is tolerant to pollution [68], and can impact aquaculture through fouling [15, 69]. Molgula manhattensis has been reported in the Mediterranean [71], western US [72], Argentina [73], Japan [74], Australia [75], and China [76], and it is thought to have been introduced to these locations via hull fouling and oyster translocations [74, 77]. Perophora viridis was first described in New England (northeastern US) [54], and since then, it has been observed all the way down to Florida, the Caribbean Sea, Brazil, Spain, and Italy [49, 78–85]. Although the species is widespread where it has been observed, little is known of its tolerance thresholds.

The most relevant environmental variables associated with ascidian distribution are seawater temperature and salinity [41, 86–88]. However, widely distributed and invasive species are notorious for their tolerance of a wide range of salinity and temperature [89–94]. All farms visited herein were in shallow marsh habitats where sharp changes in salinity and temperature are common (i.e., temperatures from 9 to 30°C and salinities from 26 to 38.5 [88]), and accordingly, only widely distributed species were observed. In addition, while shellfish farms have dedicated boats that rarely leave the facility, marinas experience constant vessel traffic. Thus, lower species richness (SpR) and abundance in shellfish farms than in marinas may result from harsher environmental conditions in shallow marsh habitats and lower propagule pressure. The wide distribution and successful introduction of the five species observed in shellfish farms into many regions of the world, and their tolerance to wide fluctuations in salinity and temperature suggests these animals can survive in different environments through adaptive evolution [95]. For M. manhattensis, Chen et al. [95] suggested that local environmental factors, notably the salinity-related variables, were crucial evolutionary forces in driving adaptive divergence. For S. plicata, Galià-Camps et al. [96] found large inversions in four chromosomes that were enriched with genes thought to increase the fitness of animals in estuary and harbor environments.

5. Conclusion

Highly adaptable species are more difficult to eradicate, and thus, current efforts focus on mitigation. Flipping cages remains the easiest, most cost-efficient, and most common method used [31, 32], with frequency being key to successful fouling control. For instance, while most farms flipped cages every 2–3 weeks, the UNCW Shellfish Hatchery did it weekly (Wilbur, pers. comm.), resulting in the lowest ascidian abundance recorded in this study. Although the increased frequency of flipping cages appeared to reduce ascidian fouling, it may not be feasible at other farms since the UNCW Shellfish Hatchery relied on students to perform this task. Relatedly, regular maintenance of biofouling organisms on gear could have led to an underestimate of ascidian abundance herein, since the timing and frequency of flipping cages differed among the visited farms. To understand the full implications of ascidian growth on shellfish farming, further studies should consider cage maintenance schedules and conduct seasonal surveys to monitor changes in ascidian assemblages over time.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Jordan Pilcher and Grace Monteith equally contributed to this work.

Funding

This research was funded by a UNCW CSURF Research Supplies Award for Undergraduate Research to Jordan Pilcher, a Wrightsville Beach King Mackerel Tournament Undergraduate Research Fellowship to Grace Monteith, and a Ralph W. Brauer Endowed Fellowship to Brenna Hutchings.

Supporting Information

Additional supporting information can be found online in the Supporting Information section.