**About the Editor**

#### **Francesco Tiralongo**

Francesco Tiralongo is an ichthyologist at the University of Catania and has authored numerous international peer-reviewed scientific articles and conferences on Mediterranean fishes. He is an avid explorer of coastal aquatic habitats and is mainly interested in the study of coastal fish species. His current research focuses on coastal fish biology and ecology, fisheries, and non-indigenous species in the Mediterranean Sea.

### *Editorial* **Coastal Fish Research**

**Francesco Tiralongo**

Department of Biological, Geological and Environmental Sciences, University of Catania, 95124 Catania, Italy; francesco.tiralongo@unict.it

Coastal fish are key components of marine ecosystems, influencing, directly or indirectly, marine life worldwide. Furthermore, among coastal fish, there are many species that represent target species for many fisheries (commercial and recreational) and contribute considerably to the economy of several coastal countries. Despite this, the biology and ecology of several species still remain little known or unknown, with several species waiting to be described. Furthermore, many fish stocks are being overexploited and in urgent need of sustainable management. Another interesting side of coastal fish is their importance as bioindicators of the status of the aquatic ecosystems. Indeed, often, coastal fish are directly exposed (and sometimes seriously threatened) to a variety of human impacts (e.g., pollution, habitat destruction, overfishing) [1]. On the other hand, invasive alien coastal fish represent in many areas a serious threat to marine biodiversity and in some cases to economy and human health.

It thus appears clear that understanding and deepening knowledge around biology and ecology of coastal fish is one of the main challenges for marine biologists and ecologists. For example, in an era in which invasive alien species threaten marine biodiversity, but can also have economic and human health impacts, the understanding of the dynamics and processes underlying biological invasions is of fundamental importance for effective management and conservation of biodiversity and ecological integrity [2]. Furthermore, the increasing marine pollution represents a serious threat to coastal fish and to marine life in general, although this phenomenon encompasses a great variety of aspects and effects on marine fish are often difficult to detect and quantify. However, some types of laboratory analysis on coastal fish fauna can give good indications of the general status of the marine environment and of the effects that specific pollutants can have on fish populations.

This special edition is intended to be a contribution to the knowledge of several aspects of fish biology and ecology: coastal fish diversity, coastal fisheries and commercial species, biological invasions process and non-indigenous species, interactions between fish and their environment, ethology, bioaccumulation and reproduction [1–12]. I hope this special issue will be helpful for all marine biologists involved in studies on fish biology and ecology.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The author declares no conflict of interest.

**Citation:** Tiralongo, F. Coastal Fish Research. *J. Mar. Sci. Eng.* **2021**, *9*, 546. https://doi.org/10.3390/jmse905 0546

Received: 12 May 2021 Accepted: 17 May 2021 Published: 18 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **References**


## *Article* **Invasive Species Control: Predation on the Alien Crab** *Percnon gibbesi* **(H. Milne Edwards, 1853) (Malacostraca: Percnidae) by the Rock Goby,** *Gobius paganellus* **Linnaeus, 1758 (Actinopterygii: Gobiidae)**

**Francesco Tiralongo 1,2,\* , Giuseppina Messina <sup>1</sup> and Bianca Maria Lombardo <sup>1</sup>**


**\*** Correspondence: francesco.tiralongo@unict.it


**Citation:** Tiralongo, F.; Messina, G.; Lombardo, B.M. Invasive Species Control: Predation on the Alien Crab *Percnon gibbesi* (H. Milne Edwards, 1853) (Malacostraca: Percnidae) by the Rock Goby, *Gobius paganellus* Linnaeus, 1758 (Actinopterygii: Gobiidae). *J. Mar. Sci. Eng.* **2021**, *9*, 393. https://doi.org/10.3390/ jmse9040393

Academic Editor: Gualtiero Basilone

Received: 22 February 2021 Accepted: 2 April 2021 Published: 7 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Abstract:** Invasive alien species (IAS) are one of the greatest causes of native species extinction. Indeed, they represent a global threat for biodiversity and can also affect the economy and human health. The colonization success of IAS is presumably not only due to their biological and ecological characteristics, but also to the lack of predators and/or parasites in the invaded new areas. In the present work, we demonstrate evidence of predation of the invasive alien crab *Percnon gibbesi* (H. Milne Edwards, 1853) by the Rock Goby *Gobius paganellus* Linnaeus, 1758. The diet of *G. paganellus* was studied analyzing the stomach content of 162 specimens collected in the central Mediterranean Sea. The results obtained from the calculation of the diet indices, namely, frequency of occurrence (%F), percentage weight (%W), percentage abundance (%N), and the Index of Relative Importance (%IRI), showed that small benthic crustaceans were the main prey types. Additionally, these indices and the Levins' index (B*i*) clearly indicated that the invasive crab *P. gibbesi* was by far the most abundant prey type in the diet of *G. paganellus*. The relevance of this predator–prey interaction and the role of native species for the biological control of invasive ones are discussed. We also provide a general view on the diet of *G. paganellus* and other biological and ecological aspects of specimens studied from the central Mediterranean Sea.

**Keywords:** Mediterranean Sea; invasive species; non-indigenous species; biological control; prey–predator interactions

#### **1. Introduction**

Non-indigenous species (NIS), and in particular invasive alien species (IAS), represent a serious threat to ecosystems' integrity, interfering with key ecological processes. Several studies have demonstrated that IAS can reduce the abundance and presence of native species through predation or competition and can alter food webs and community structure [1,2]. Indeed, they are considered to be the greatest cause of native species extinctions after habitat destruction [3]. In some cases, these alterations to the ecosystem can cause severe economic losses, sometimes exceeding those of natural disasters, and represent a threat to human health [4,5]. Although the dynamics of biological invasions are complex and often unclear, the results of several studies have suggested that areas with high species richness are more resistant to biological invasions than areas poor in species. In the former areas, the scarce availability of a free ecological niche represents an obstacle to the spread of IAS, reducing the possibility of settlement and/or expansion. This hypothesis is known as the "biotic resistance hypothesis" [6,7]. In this context, but also in general, the role of native predators can be relevant for the biological control of IAS. Indeed, in some cases, native species can have a high potential to exert predatory control on invasive ones [8]. Among these, coastal fish species can potentially play a key role in the predation of IAS.

Gobies comprise more than 1900 species, and are the largest family (Gobiidae) of marine fishes [9,10]. In the Mediterranean Sea, this family includes 73 species, thus being the most diverse fish group of the region [11]. Despite this and their relevance for marine ecosystems, an increasing interest in gobies only started recently, with several published studies on their biology and ecology and the description of new species [12–18].

*Gobius paganellus* Linnaeus, 1758 is a medium-sized goby (Gobiidae), whose distribution extends from the Mediterranean and Black Sea to the eastern Atlantic (from Scotland to Senegal), including the Atlantic islands of the Azores, Madeira, and the Canaries [19]. Furthermore, it represents one of the few fish species that has performed an anti-Lessepsian migration, reaching the Red Sea through the Suez Canal [20]. The maximum reported total length (TL) is 14 cm for males and 14.3 cm for females [12], but common sizes range from about 8 to 12 cm TL, and no marked differences in size are present between sexes [12,21,22]. It is a shallow waters species generally common on rocky bottoms, often occurring in tidal pools [22,23]. Total length at first maturity was estimated at 5.2–11.4 cm TL, depending on the sex and location, a size that can be reached at 1–3 years of age [12,21,22,24,25]. Crustaceans are the main prey types in the diet of *G. paganellus* [12,21,24,26], and, depending on the size, area, and season, there is a variation in the dominance of the different species on which this goby feeds. Hence, *G. paganellus* is an opportunistic predator. However, among crustaceans, amphipods and isopods, followed by Caridea and Brachyura, were recorded to be the most common prey in the diet of the species [12,21,24,26].

*Percnon gibbesi* (H. Milne Edwards, 1853), commonly known as the Nimble Spray Crab, is a medium-sized crab whose distribution extends from California to Chile in the Pacific Ocean, from North Carolina to Brazil in the Western Atlantic Ocean, and from the Azores to Angola in the Eastern Atlantic Ocean [27]. After its first record in 1999 in the Mediterranean Sea, *P. gibbesi* has undergone a rapid expansion in the whole basin [28,29]. By year 2000, the rapid spread of the species was recorded in Sicily [30]. The great colonization success of this crab was attributed to several factors, such as an increase of sea water temperatures, the absence of competitors, the availability of unoccupied ecological niches, and life history characteristics such as some aspects of its reproductive biology and long planktotrophic larval stage [31]. However, to the best of our knowledge, the predation of this species by a native species has never been demonstrated for the Mediterranean Sea, because *P. gibbesi* was never found in the stomach of any analyzed species and no predators were directly observed to prey on the crab (e.g., [7,12,15,21]). Yet, native species can prey on invasive ones, and, in some cases, contribute to controlling their populations [8,32–36]. Although several means of introduction have been hypothesized for *P. gibbesi* (i.e., aquarium release, larval drift through the Strait of Gibraltar, adult migration, ballast waters, ship hulls), the most likely appears to be the introduction through shipping [29,37,38]. This species was often found among boulders covered by algal mats or in which this covering was almost absent, in very shallow waters [29]. This habitat simultaneously provides protection from predators and food supply, represented by small algae and other small sessile organisms [30]. *Percnon gibbesi* has been considered mainly herbivorous (algae-eating), but some authors demonstrated its opportunistic feeding behavior [29,30]. Indeed, the species was observed to prey on hermit crabs and polychaetes, and analysis of stomach content revealed that gastropods and crustaceans can constitute a considerable part of its diet [39,40]. In order to avoid diurnal predators, *P. gibbesi* is more active starting from the evening hours [31,40]. In Mediterranean waters, ovigerous females were observed between May and September. Sexual dimorphism is present, with males showing chela length with a larger positive allometry than that of females [31]. According to some authors, *P. gibbesi* is a potential competitor of *Pachygrapsus marmoratus* (J.C. Fabricius, 1787), both for space and food resources, and a similar situation could be present with *Eriphia verrucosa* (Forskål, 1775), although both native crabs usually prefer inhabiting the rocky intertidal and very shallow waters, among crevices and holes or under boulders [31].

In the present study, we provide the first direct evidence of substantial predation of a native species, *G. paganellus*, on the invasive alien crab, *P. gibbesi*, emphasizing the role of the goby in the biological control of this invasive species. Furthermore, we also provide the first recorded data on some aspects of the biology and ecology of *G. paganellus* from the Ionian Sea (central Mediterranean Sea): length–weight relationship, total length–standard length relationship, size frequency distribution, diet, and feeding habits. For these latter two points, we analyzed and discussed the prey selectivity of the species.

#### **2. Materials and Methods**

A total of 162 specimens of *G. paganellus* were collected in the Ionian Sea (south-east coast of Sicily, central Mediterranean Sea), in an area between the localities of Avola and Noto, along a coastline extending for about 2 km (Figure 1). In order to better represent the investigated population, two collection areas were selected, that of Noto represented by a small semi natural harbor (36.86994 N, 15.13738 E) with rocky bottom and boulders and that of Avola represented by a natural rocky bottom area (36.88628 N, 15.14041 E). These areas were distant from each other by about 2 km and had a depth range of 0.1–1.5 m. Sampling was performed during daytime with calm sea conditions, between 4 September and 5 December 2020. In this period, a total of 12 samplings were carried out, with an average of 13.5 specimens per sampling day (Table 1).

The specimens were caught with a fishing rod, using as bait fresh pieces of the Deepwater Rose Shrimp, *Parapenaeus longirostris* (H. Lucas, 1846). Specimens were killed in ice water immediately after their capture. Subsequently, each specimen was weighed and measured (total length and standard length). Total length and weight measures were used for the length–weight relationship following the formula: W = aTLb, where W is the weight in grams (g), TL is the total length in centimetres (cm), a is the intercept, and b is the slope of the regression curve; when b = 3, the increase in weight is isometric; contrariwise, when the value of b is different to 3, the weight increase is allometric. In this latter case, if b > 3, the weight increase is positive allometric, and if b < 3, the weight increase is negative allometric. A one-sample t-test was used to verify the null hypothesis of the isometric growth (H0: b = 3). Length–weight relationships are valuable measurements in order to evaluate and compare the health conditions of species' population [41,42]. Total length (TL) and standard length (SL) measures were used for the total length–standard length relationship following the formula: TL = αSL + β, where α is the slope and β is the intercept of the regression line.


**Table 1.** Means of the basic measurements per sampling day for *Gobius paganellus*; N = total number of specimens; N Avola = specimens collected at Avola; N Noto = specimens collected at Noto; N Prey = specimens with at least one prey in their stomach; TL = total length; SL = standard length; W = weight.

**Figure 1.** Study areas of Noto (**in red**) and Avola (**in yellow**) in the Ionian coast of Sicily (central Mediterranean Sea).

After measurements, the stomach was removed and the content analyzed under a stereoscopic microscope. All the prey items found were counted, washed in clean seawater and dried with blotter paper, identified to the lowest taxonomic level possible using identification manuals [43,44], and weighed to the nearest 0.001 g. After examination, stomach contents and gobies were preserved in ethanol.

The frequency of occurrence—%F (defined as the number of stomachs in which each specific prey type is represented expressed as percentage), percentage weight—%W (defined as the weight of each prey type in the stomachs examined expressed as percentage), percentage abundance—%N (defined as the number of individuals of each prey type in the stomachs examined expressed as percentage), and the Index of Relative Importance— %IRI (calculated by summing %N and %W values and multiplying with %F value) were calculated for each taxon [45,46]. The percentage of empty stomachs (vacuity index)— V% (number of empty stomachs/total number of stomachs examined) × 100, was also calculated.

Prey were grouped into three categories on the basis of their percentage abundance (%N) [47]: dominant (N > 50%), secondary (10%<N< 50%), and accidental (N < 10%).

Standardized Levins' index (B*i*) was used to evaluate the breadth of the diet [48]:

$$\mathbf{B} = \frac{1}{\sum p\_j^2} \tag{1}$$

$$\text{Bi} = \frac{\text{B} - 1}{\text{B}\_{\text{max}} - 1} \tag{2}$$

where *pj* is the relative frequency specimens in the jth prey item and B*max* is the total number of prey item categories found. B*i* is comprised between 0 and 1. The higher the value of this index, the wider the trophic niche of the species will be. Hence, if B*i* < 0.40, the

species is considered a "specialist feeder", if 0.40 < Bi < 0.60 the species is considered an "intermediate feeder", and if B*i* > 0.60, the species is considered a "generalist feeder" [49].

A cumulative prey curve [50] was computed with R Studio [51] using the "vegan" package in order to evaluate whether the number of analyzed stomachs was sufficient to describe the diet of the species. The estimated number of prey groups with the associated SD were plotted against the cumulative number of individuals whose stomach was examined.

#### **3. Results**

One hundred and fifteen out of a total of 162 specimens examined of *G. paganellus* had prey in their stomachs; thus, the vacuity index (percentage of empty stomachs) was V% = 29.01. The cumulative prey curve approached an asymptote, suggesting that the analysis of about 100 not empty stomachs provides a reliable description of the diet of the goby (Figure 2). Analysis of stomach contents of *G. paganellus* included 20 prey types. Data showed that *G. paganellus* feed mainly on small benthic crustaceans (Table 2). In particular, values of %F, %W, %N, and %IRI indicated that *P. gibbesi*, which accounted for about one third of all prey items, was by far the most abundant prey type. This was also strongly supported by the Levins' index value (B*i* = 0.39), indicating a narrow trophic niche ("specialist feeder"). The carapace width (CW) of the specimens of *P. gibbesi* found in the stomachs was about or less than 1 cm. On a total of 20 prey types recorded, no "dominant" (N > 50%) prey types were found, and the only "secondary" prey type (10% < N < 50%) was represented by *P. gibbesi*. Hence, except for *P. gibbesi*, all the prey types fall into the category "accidental preys" (N < 10%) (Table 2). However, among these latter preys, the most represented were some Brachyura (*Pisidia* sp., *Xantho* sp., and juvenile specimens of *P. marmoratus*) and the hermit crab *Clibanarius erythropus* (Latreille, 1818). *Eriphia verrucosa* was the less represented crab (%F = 4.35). The other groups of crustaceans found in the stomachs of the Rock Goby were Amphipoda, Caridea, and Isopoda. These latter groups of crustaceans and Algae showed similar values in diet indices (e.g., %F = 6.09–6.96). Mollusca (gastropods) were overall less represented (%F = 0.87–1.74, excluding value for not identified Gastropoda), followed by Polychaeta and Seagrass (%F = 2.61 and 0.87, respectively) (Table 2).

**Figure 2.** Cumulative prey curve (**in red**) as a function of sample size for all stomachs analyzed of *Gobius paganellus*. Standard deviation (SD) in grey delimited by dashed black lines.


**Table 2.** Diet composition of *G. paganellus* (N = 115) from the Ionian Sea (central Mediterranean Sea). %F = percentage frequency of occurrence; %N = percentage in number; %W = percentage in biomass; IRI = index of relative importance of prey items and its percentage (%IRI). Results for *Percnon gibbesi* are underlined.

In the specimens sampled in the small semi natural harbor (N = 90), *P. gibbesi* was found in 43.3% (N = 39) of the stomachs. In contrast, *P. gibbesi* was absent from the stomach contents of specimens sampled in the nearby natural rocky bottom area. In the whole sample of *G. paganellus* with at least one prey in their stomach (N = 115), *P. gibbesi* was found in 33.9% of the stomachs analyzed. However, in order to compare the diet of the goby in the aforementioned locations, removing *P. gibbesi* from the analysis did not find any significant differences between them. In all cases, crabs and hermit crabs represented the most common prey types of the species, although no clear dominance was evident for any prey type (with the exception of *P. gibbesi*).

The size frequency distribution of the sampled specimens (N = 162) showed a size range of 7.1–11.6 cm TL, with a mean of 9.5 cm (Table 3). The size frequency distribution was multimodal (Figure 3). The total length–weight relationship showed an isometric growth (b = 2.959; *p*-value > 0.05) (Table 3), while the parameters of the linear regression of total length–standard length relationship were: TL = 1.136SL + 0.4822 (Figure 4). Means of the basic measurements per sampling day are reported in Table 1.

**Table 3.** Total length, standard length, weight, and total length–weight relationship parameters of *G. paganellus* in the Ionian Sea (central Mediterranean Sea); a = intercept of the regression curve; b = slope; r2 = coefficient of correlation; C.I. = 95% confidence interval.


**Figure 3.** Size distribution (TL in cm) of sampled specimens of *G. paganellus*.

**Figure 4.** Total length–weight relationship (**A**) and total length–standard length relationship (**B**) of *G. paganellus*.

#### **4. Discussion**

The diet analysis of *G. paganellus* clearly showed the dominance of the alien crab *P. gibbesi*. Indeed, this species of crab was found in the stomach of a considerable percentage of specimens (%F = 33.91; %IRI = 77.01). Considering the small size of the crabs found in the stomachs, they were all juveniles. A noteworthy fact is that *P. gibbesi* was found only in the stomach of specimens sampled in the small harbor (N = 90). This can be explained by the fact that gobies are usually generalists and opportunistic feeders that take advantage of the most available prey. Furthermore, the gobies' diet was found to be often dependent on area and season [14,15,52]. Based on this, the relative abundance of alien crabs found in the stomach of specimens sampled in the small harbor can be the direct result of the different abundance of *P. gibbesi* in the two areas investigated, and point out the preference of the invasive crab for areas characterized by the presence of breakwaters and/or boulders. Indeed, some authors demonstrated the preference of the invasive crab for harbor's breakwaters, where diversity levels are generally low and where it can find a suitable habitat among breakwaters and boulders [39]. However, from our data, it was not possible to determine whether the goby selectively preys on the crab, or if the abundance of *P. gibbesi* found in the stomachs of *G. paganellus* was related to the greater presence of *P. gibbesi* in the waters of the small harbor.

In all cases, despite the relatively high value of the frequency of occurrence obtained for *P. gibbesi*, the overall diet of *G. paganellus* included a quite wide variety of benthic invertebrates, mainly consisting of small benthic crustaceans. Hence, the Rock Goby may be generally considered as an opportunistic carnivorous species. In other words, the narrow trophic niche indicated by the Levins' index is the result of the abundance of *P. gibbesi* in the stomachs of specimens of *G. paganellus* from the small semi natural harbor, otherwise *G. paganellus* can be considered as a "generalist feeder", as usually reported in literature. After *P. gibbesi*, other crab species, such as *Pisidia* sp., *Xantho* sp., and *P. marmoratus* (juveniles) and the hermit crab *C. erythropus* represent the most important prey types of *G. paganellus*. On the other hand, juveniles of *E. verrucosa* were less represented. Other relatively well represented groups of crustaceans were Amphipoda, Caridea, and Isopoda. Gastropods and polychaetes were rarely found. Finally, algae and seagrasses were probably accidentally ingested, as suggested by some authors for algae [24]. Our results are in general agreement with those reported in literature [12,21,24,26] that describe the diet of *G. paganellus* as mainly composed of small benthic crustaceans, although with some differences among the crustacean groups recorded.

Considering the size range of the analyzed specimens, all the studied individuals of *G. paganellus* were of medium and large size. Indeed, no individual smaller than 7.1 cm TL was recorded. The total length–weight relationship showed an isometric growth, while a positive allometric growth was recorded in specimens from a coastal lagoon of Spain [53]. The analysis of the total length–standard length relationship demonstrated that the SL is on average 83.16% of the TL.

Our study demonstrates for the first time how a native fish species can effectively prey on this invasive crab, reducing the abundance of the established population. Although *P. gibbesi* is quite elusive, *G. paganellus*, a strictly benthic goby of rocky and mixed bottoms, is able to predate on it among rocks and boulders, especially in areas in which the crab is particularly abundant and probably other resources are scarce. Further studies on a large spatio-temporal scale and on the ecology and mechanisms of predation and prey selection are necessary to better understand the role that can have predators such as *G. paganellus* and other native intertidal and shallow waters fish species on the Mediterranean population of *P. gibbesi*. Furthermore, considering the sizes of the preys found and the mouth opening of *G. paganellus*, a medium sized species of goby, the species is able to prey only on juvenile specimens of the crab, while species of larger size and with a wider mouth opening (e.g., *Gobius cobitis*, *Muraena helena*, *Scorpaena maderensis*, *Scorpaena porcus*, *Serranus cabrilla*, *Serranus scriba*) should be investigated for their ability to feed also on adult specimens of *P. gibbesi*. In this regard, it is interesting to note how Sciberras and Schembri [31] reported

that in the nearby Maltese waters, the juveniles of *P. gibbesi* were observed between the end of September until at least March, a period that overlaps with that of our study. This would suggest how in Sicily the recruitment could also occur in this period and probably extend for a similar period too. Further studies mentioned above could also clarify this point. To further confirm what was also reported by the same authors, namely the habitat overlap between *P. gibbesi* and *P. marmoratus* in particular, but also with *E. verrucosa*, we also found these two latter species in the stomach content of *G. paganellus*, and *P. marmoratus* was more common than *E. verrucosa*. Another interesting aspect concerning the native predators' control of the population of the invasive *P. gibbesi* was recently pointed out by Noè et al., 2018 [7]. Results obtained by the authors suggested how in areas with high diversity and an abundance of potential predators (e.g., marine protected areas) of *P. gibbesi*, the abundance of the invasive crab is generally lower than in non-protected areas where potential predators are scarcely represented. Other studies are needed in order to clarify the relationships and dynamics between *P. gibbesi* and the presence and abundance of its actual predators.

Invasive species can alter marine ecosystems and cause biodiversity loss, economic damages affecting areas such as fisheries, tourism, and recreational activities, and a threat to human health. Especially in marine environments, totally eradicating or stopping the spread of an invasive alien species have never been successful, and the only valid alternative to date is to limit the spread and abundance through targeted actions [3–5]. For some palatable or even edible alien species, direct actions such as fisheries for human consumption can be a valid solution in order to reduce population. In contrast, when we face non-edible invasive species such as the case of *P. gibbesi*, the promotion and protection of natural predators of invasive species, and the general promotion of the "biotic resistance" (that is, the ability of native species in a community to limit and control the invasion of other species), can be valid additional resources in order to control invasive populations of alien species [7,54,55]. Several studies have already shown how some native species prey on non-indigenous ones, and, in some cases, they are capable of effectively controlling their populations [8,34–36]. Although further studies are necessary in order to better understand this and other predator (native)–prey (IAS) interactions, *G. paganellus* seems to perform the function of "autoregulator" of *P. gibbesi* abundance. Indeed, considering the opportunistic and generalist feeding behavior of *G. paganellus* (and of gobies in general), it preys on the crab especially when the latter is very abundant.

Invasive species can replace or limit the abundance of native ones, alter the habitat structure, and interfere with biogeochemical processes [8,56]. As mentioned above, in a marine environment, the eradication of IAS is complicated, and no eradication attempts have so far been successful. Hence, prevention methods should be prioritized over those of eradication. In this regard, the "biotic resistance" provided by a well-structured native community can act as a natural barrier in preventing or slowing biological invasions. Indeed, on the basis of the mechanisms of competition, IAS cannot establish in a niche similar to that of native species [57]. Furthermore, the occupation of niches is more complete in a species-rich and well-structured native community, and this further contributes to preventing biological invasions [58]. In this regard, the role of native predators, such as in the case here presented of *G. paganellus*, can effectively contribute to controlling populations of invasive species [8]. Although we have no data to state if some specimens of the Rock Goby can learn to prey selectively on the invasive crab, they can certainly contribute to reducing their number. Furthermore, the discovery of the existence of a combination of native predators of the crab would show how, overall and at least in some locations, they could significantly contribute to controlling and limiting the population's growth of the invasive crab. Particular measures could also be promoted in order to protect the population of key native species from human activities.

#### **5. Conclusions**

In conclusion, our study demonstrates how the native fish *G. paganellus* can prey on the invasive crab *P. gibbesi*, being able to contribute to the control of the population of this IAS in the Mediterranean Sea. Although *P. gibbesi* was the dominant prey type, the diet of *G. paganellus* was composed of a wide variety of invertebrates, mainly represented by small benthic crustaceans, brachyurans in particular. Hence, we can consider this species as an opportunistic feeder able to prey on *P. gibbesi* too, thus potentially playing a role in the biological control of the species. However, more studies are needed to understand if the native goby can (and under what conditions) develop the ability to selectively feed on *P. gibbesi*. Furthermore, other studies can help to expand this research to other species and areas in order to evaluate the overall impact of predation of the coastal fish community on this invasive crab (and other invasive species), and relate the presence of the major predators of *P. gibbesi* to its abundance levels at several locations. This study also provided the first biological and ecological data on *G. paganellus* from specimens from the Ionian Sea (central Mediterranean Sea).

The lack of predators and/or parasites can cause overpopulation of invasive species in their new colonized areas, especially if these species cannot be exploited economically by fishery because they have no economic value. *Percnon gibbesi* falls in this latter category and showed a great success in colonizing the Mediterranean Sea, with potentially negative although unpredictable effects on the ecosystem.

To date, no studies had shown a direct predation on this crab by a native species. Although our study clearly demonstrated the predation of the native *G. paganellus* on the invasive crab *P. gibbesi*, further studies are still needed in order to better understand the mechanisms of this predator–prey interaction, the degree of contribution that this predation can have in controlling the abundance of this invasive species, and if other Mediterranean populations of the goby have adapted or are able to prey on this invasive crab. Furthermore, it is very likely that other species as well, such as some coastal fish, prey on *P. gibbesi*, and other studies should focus on finding out which ones and to what extent they can contribute overall to the control of the invasive Mediterranean population of the crab.

**Author Contributions:** Conceptualization: F.T.; methodology: F.T., G.M. and B.M.L.; investigation: F.T. and G.M., formal analysis: F.T., G.M. and B.M.L.; writing—original draft preparation: F.T. and G.M.; writing—review and editing: F.T., G.M. and B.M.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Ethical review and approval are not necessary for this study because collected specimens come from fishing activities and have been treated ethically.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data will be made available on request.

**Acknowledgments:** Authors are grateful to Giacomo Bernardi, Department of Ecology and Evolutionary Biology, University of California Santa Cruz, for the English language review, and to the three anonymous reviewers for their helpful comments.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Article*

## **Relative Influence of Environmental Factors on Biodiversity and Behavioural Traits of a Rare Mesopelagic Fish,** *Trachipterus trachypterus* **(Gmelin, 1789), in a Continental Shelf Front of the Mediterranean Sea**

#### **Armando Macali 1,\*, Alexander Semenov <sup>2</sup> , Francesco Paladini de Mendoza 3, Alessia Dinoi <sup>4</sup> , Elisa Bergami <sup>5</sup> and Francesco Tiralongo 3,6**


Received: 7 July 2020; Accepted: 30 July 2020; Published: 2 August 2020

**Abstract:** Coastal environments can be influenced by water body masses with particular physical, chemical, and biological properties that create favourable conditions for the development of unique planktonic communities. In this study, we investigated a continental shelf front at Ponza Island (Tyrrhenian Sea) and discussed its diversity and complexity in relation to major environmental parameters. Moon phase and current direction were found to play a significant role in shaping species abundance and behaviour. During in situ observations, we also provided the first data on the behaviour of juveniles of a rare mesopelagic species, *Trachipterus trachypterus*, suggesting the occurrence of Batesian mimicry.

**Keywords:** Trachipteridae; Ponza Island; upwelling; plankton diversity; Batesian mimicry

#### **1. Introduction**

Open water environments are highly dynamic ecosystems [1], with ecological processes spreading over a wide range of spatial and temporal scales [2]. At a meso- and sub-mesoscale (~1–100 km), these processes emerge as dominant structuring regimes for populations of marine organisms in the short time span (days to months) [3,4], encompassing key biological dynamics, such as bloom lifetime or behavioural switches of marine predators [5]. These ecological processes are particularly relevant for drifting organisms (plankton) that are advected and dispersed by the ocean currents [6]. Within this complex scenario, sampling probabilities for uncommon and little-known species can be higher than those reported in all other marine environments, in which these species are typically rare and occur at low abundances. This may result in uncertainty about the real species abundances and their ecological traits. As a result, modern biodiversity surveillance programs often investigate the occurrence of species mostly present at moderate-to-high abundance level [7,8].

The complex interactions between coastal morphology and sea surface circulation of the Mediterranean Sea promote a high degree of ecosystem patchiness [9]. The environmental characteristics (e.g., salinity and temperature), as well as the dynamics of enrichment processes in the water column [5], lead to differences in distribution and abundance of pelagic species [10–12]. While marine macro-ecology has benefited from the analysis of spatially extensive data sites, inferences about ecological processes are better evaluated by dynamic data collection over temporal scales on a smaller geographic range [13]. Animal behaviour also affect assemblage composition, as for species displaying avoidance behaviour (e.g., due to increased predation risk) or with small home ranges, which are less likely to be observed than schooling or highly mobile species [14].

Within the Mediterranean fish biodiversity, little is still known on mesopelagic communities. Among deep water species, representatives of the family Trachipteridae Swainson, 1839 (order Lampriformes) are rare encounters. They are represented by 10 species distributed in 3 genera [15]: *Desmodema* (Walters and Fitch, 1960), *Trachipterus* (Goüan, 1770), and *Zu* (Walters and Fitch, 1960) [16]. Of these, only *Trachipterus arcticus* (Brünnich, 1788), *Trachipterus trachypterus* (Gmelin, 1789) and *Zu cristatus* (Bonelli, 1819) have been reported in the Mediterranean Sea. The former is a North Atlantic species, with a single record in the Mediterranean Sea from Spain [17]. The other two species, *T. trachypterus* and *Z. cristatus*, reported from tropical and temperate waters of all oceans, have been found in several Mediterranean areas [18–27]. In the Mediterranean Sea, fish of this family are accidentally caught with professional fishing gears, but always in low numbers [20,25,28]. Given the complexity of mesopelagic environments, an understanding of the triggers for the distribution of rare mesopelagic species requires a more comprehensive knowledge of the temporal pattern of environmental variation in a variety of timescales. For this reason, long-term environmental monitoring at aggregation sites is of critical importance.

With the present study, we examined the extent to which key features of the hydrodynamic environment predict the presence of *T. trachypterus* in a Mediterranean continental shelf front over a 21-days timescale. Using multivariate hierarchical analysis, we examined the temporal changes in species occurrence at an aggregation site. Field observations over time have allowed us to discuss on the drivers affecting the occurrence at particular times and on some relevant ecological and behavioural traits of these species.

#### **2. Materials and Methods**

#### *2.1. Study Area and Sampling Design*

The study was performed in the framework of the AQUATILIS EXPEDITION at Ponza Island (Tyrrhenian Sea, Italy) from the 8th to the 28th of April 2018. Due to its bathymetric profile, the area is exposed to prominent surface and upwelling filaments, resulting in a complex hydrodynamism that favours the accumulation of planktonic species in patched areas, according to the prevalent weather forcing [29]. Diving sites were close to "Secca delle Formiche" (40.884947◦ N, 12.978043◦ E), a group of emerged rocks, located 0.8 nautical miles (nm) SE from the port of Ponza. The bank rapidly sinks from a depth of 5 m to over 500 m (Figure 1), representing a suitable place to study the upwelling of deep mesopelagic species.

Previous surveys conducted in April 2016 and 2017 highlighted a remarkable difference in plankton abundance and biodiversity in the study area according to time; in particular, observation during night dives showed a more complex plankton biodiversity, if compared with day dives (authors' personal note). As a consequence, in the present study, scuba night dives have been planned to start one hour after sunset. Two different groups of three divers each equipped with Nikon D850 with 50 mm f2 Zeiss Milvus macro lens for photography, Panasonic Lumix GH5 with Panasonic Lumix G 8 mm f3.5 fish eye and Panasonic Lumix 15 mm f1.7 lenses for video and tools (fine mesh hand nets, 250 mL plastic cans) for sampling collection, performed alternate dives. In order to increase the image quality, all the cameras were equipped with a buoyancy stabilization system. Sampling depth varied

between 0.5 and 10 m, freely drifting within the surface current. To ensure taxon identity, 4 specimens of *T. trachypterus* were collected and analyzed for morphological traits. Behaviour was recorded by filming all the specimens detected. Observations followed the same schedule, starting one hour after sunset, freely drifting in the first 10 m of the water column. Surveys were conducted daily between 21:00 and 24:00 h.

**Figure 1.** Regional view of the coastal site with the localization of the study area (Ponza Island, within the Pontine Island archipelago) and weather station used for the physical process analysis.

#### *2.2. Environmental Data Collection*

Main abiotic environmental conditions were inferred from numerical models and in situ measurements (current direction). To this aim, wind records from available meteorological station and numerical results of Surface Sea Temperature (SST) and Salinity (SS) were collected considering both surveying period and long-time series. The wind records were downloaded from the Gaeta Port Authority and the Mareographic network websites (www.mareografico.it; www.portidiroma.it). Ponza's records extend only for a 4-year period (2011–2015), while more than 10-year records are available from Gaeta (2007–2018) (Supplementary Materials, Figure S1). Despite the distance between stations, wind roses can be considered comparable [29,30]. During surveys, we used Gaeta Meteo Station period to analyse wind conditions. The SST and SS datasets were downloaded from the "Copernicus" data portal (http://marine.copernicus.eu, accessed on the 21st of November 2018). SST and SS from 1987 to present were analysed, producing long-term temporal mean distribution of variables during April months and mean conditions during the surveyed period.

Moonlight is known to affect zooplankton and micro-nekton dynamics, as well as their predators in tropical, subtropical, and Arctic waters [31–36]. Moonlight also drives vertical migrations to bathypelagic depths (~1000 m) involving a cascade response, with deeper-living organisms responding to movements of shallower-living, surface-synchronized populations [37]. According to this evidence, the main macro-zooplanktonic species composition was recorded and correlated to moon phases and moonlight intensity.

#### *2.3. Data Analysis*

Non-parametric multivariate analyses were performed to assess the relation between the species observed and the environmental parameters over the survey time period. To analyze the environmental conditions (SST, moon phase, wind direction, and current direction), the parameters were normalized and standardized to carry out a Principal Component Analysis (PCA) with Statistical Package for Social Science (SPSS, Version 26.0, IBM Corp© 2019). Once the significant environmental patterns were established, standardized data of the observed species were used to investigate the variability of these patterns. To assess variations in the species diversity, data of the thirty-five taxa were analyzed

using distance-based permutational multivariate analysis of variance (PERMANOVA) [38,39] with the method of permutation of the residuals under a reduced model, according to a 2-factor nested design: moon phase and current direction. The homogeneity of multivariate dispersions was tested with the PERMDISP routine, mainly to estimate dispersion at the moon phase level. Non-parametric multidimensional scaling ordination (nMDS) was used to examine the behaviour of the species grouped according to the current direction pattern. The Similarity Percentage analysis procedure (SIMPER) was used to identify the main species that characterized each current direction pattern. These analyses were performed on Primer v.7 with PERMANOVA+ software [39,40]. A PCA with supplementary variables was performed with CANOCO 5 [41] in order to investigate the species response to each variable. Finally, the hierarchical cluster analysis was applied to estimate the number of clusters using Primer v.7 [40].

#### **3. Results**

#### *3.1. Oceanographic Landscape*

Available time-lapse records of Ponza Island and Gaeta showed three main directions (North-East, South-East and West-Northwest) of wind. Between the 12th and the 18th of April 2018, a moderate north-west wind dominated, while in the other days, weak northern flows (8th–11th of April) and eastern flows (19th–28th of April) were recorded (Supplementary Materials, Figure S2B). The SST mean distribution of April showed a quite uniform temperature at a regional scale, with variations of less than 1 ◦C (16.8–17.6 ◦C). Between the 9th and the 18th of April 2018, the SST was generally cooler than the mean long-term map, with a weak NW-SE increasing gradient. In correspondence of the study area, the SST reached 15.3–15.5 ◦C. After the 18th of April, a sensitive increase (more than 2 ◦C) in SST occurred at a regional scale (Supplementary Materials, Figure S2A). SS mean distribution map (Supplementary Materials, Figure S2C) shows the effect of continental waters (Volturno River) which reduced SS in the nearshore. In the study site, the SS reached 38 psu on the 9th–11th of April 2018, slowly decreasing until the end of the survey (Supplementary Materials, Figure S2C).

#### *3.2. Assemblages*

A total of 35 families of meso- and macroplankton taxa has been recorded over the whole studied period, together with the associated environmental factors (Supplementary Materials, Table S1). The species list included pelagic predators (*Forskaliidae*, *Ommastrephidae, Pelagidae*, and *Phronimidae*) as well as planktivorous species (*Alciopydae*, *Fritillariidae*, *Leucotheidae*, *Mysidae*, and *Salpidae*) representing exploiting food characteristics of the current fronts. Details of the trophic and reproductive behaviour of these species are reported in the Supplementary Materials (Plate I-III).

#### *3.3. Role of Environmental Factors*

The PCA model computed with SPSS explained more than 90% of the variability for the considered environmental variables (Figure 2). Based on these results and the PERMANOVA, two environmental patterns were retained as main factors: the moon phase (*p*-value = 0.0011) and the current direction (*p*-value = 0.0015). The SST did not play a significant role in species composition and diversity, while showing high variability (range 14.5–19 ◦C) in the surveyed period.

Moonlight seems to rule the occurrence and abundance of plankton in the surface layer of the water column, with a clear increase in the occurrence of 12 taxa, consequently to the extremes of the moon phase (see Supplementary Materials, Figure S3). With the exception of Cestidae, all the other taxa included in this group showed a negative correlation to moonlight, as for Nereidae and Sepiolidae, which was also correlated to the record of mating behaviour (see Supplementary Materials, Plate I-II). Juvenile and larval stages of benthic and benthopelagic species, as Pleuronectiformes, Phycidae, Anguilliformes, and Scyllaridae, also negatively responded to moonlight.

**Figure 2.** Principal component analysis (PCA) of the environmental factors (performed with SPSS).

According to the observed pattern, the most abundant taxon during the main Eastern flow direction was represented by Forskaliidae, while in the least North-Western flow the most abundant taxon was represented by Alciopidae.

The PCA model performed with CANOCO 5 shows correlations among moon phase, current directions, and taxa. It is possible to observe how most taxa took the opposite direction as the moon phase progressed and how the main current blows eastward (Figure 3a). Arrows indicate the direction of the taxa at increasing values of the environmental variables, with longer vectors representing a greater range of variation in the observed values (Figure 3b). A stronger correlation is associated to more acute angles between vectors and the axes. The x axis explains 57.6% of the variation among parameters at each taxon, whereas the y axis explains 47.1% (Figure 3a,b).

**Figure 3.** PCA (**a**) of the encountered taxa and the two main environmental factors of moon phase and current directions. PCA with arrows (**b**) highlights the direction of the taxa at increasing values of the environmental variables (performed with CANOCO 5).

The hierarchical cluster analysis performed with Primer v7 shows 5 main clusters based on the taxa presence and the current direction as environmental factor (Figure 4).

**Figure 4.** Dendogram plot performed by Primer v7 combined with the shadow plot for the presence (from 0 to 1) in the monitored time and the daily current direction.

According to the statistical analyses on the relation between environmental constrains and species diversity, the contribution of the eastern currents in shaping plankton communities is well supported (see Figure 4). Lowest level of plankton diversity is related to north western stream, which can be considered local, as the diving site results covered by the island from this direction (0.8 nm off the bank). Species diversity mainly reflects typical pelagic assemblages, whereas the occurrence of emergent zooplankton (Amphipoda, Nereidae) and more benthic species (Octopodidae) can be considered mainly influenced by the contribution of the nearby shallow water banks ("Secca delle Formiche") and affected by moon phase rather than current direction. The occurrence of other larval and juvenile stages, as for Anguilliformes, Phycidae, Pleuronectiformes, and Scyllaridae (Figure 4), showed a clear convergence in the contribution of current direction (Figure 4) and moon phase (Supplementary Materials, Figure S3), which may be related to a shared behavioural form of detection avoidance.

#### *3.4. Behavioral Traits of T. trachypterus*

Over the whole study period, a total of 18 juveniles of *T. trachypterus* were observed, photographed and filmed during SCUBA dives at night between the 16th and the 28th of April 2018. Prolonged observations (>2 min) of the specimens were possible only for 10 fish, given the high avoidance behaviour of the species. Juveniles were observed swimming between 0.5 and 7 m below the surface, with the body obliquely or almost vertically pointed with the head towards the surface. Locomotion was performed through undulating movements of the long dorsal fin only, except for the anterior prolonged dorsal fin rays that showed limited oscillating movements (Supplementary Materials, Video 1–10). A faster escaping movement was displayed using the whole body, as showed in Supplementary Materials, Video 8. Pelvics and caudal fin remained spread, but substantially motionless or with small and occasional "snap" movements. While dorsal and pectoral fin membranes were entirely translucent, those of the wide pelvic and caudal fins showed orange blotches, with rays protruding far over the fin membrane. In some cases, juveniles oriented the dorsal or abdominal surface towards the observer, greatly limiting the area exposed to the view of the observer itself (Figure 5C,D; Supplementary Materials, Video 5–7, 9–10). At the same time, caudal and pelvic fins were maintained orthogonal to the observer, with a significant torsion of the caudal fin up to 90◦ angle (Figure 5C). This latter behaviour was displayed for a prolonged time.

**Figure 5.** Lateral (**A)**, fronto-lateral (**B**), dorsal (**C**), and frontal (**D**) view of juvenile specimens of *T. trachypterus,* with detail of fins orientation accordingly to the observer visual axis.

Fish were also observed rapidly protruding their jaws, probably preying on small planktonic organisms (Supplementary Materials, Video 4), that were very abundant in the surface water column in which specimens were observed.

Four specimens were caught by hand net and preserved in alcohol for laboratory analysis. All the main morphometric parameters are reported in Supplementary Materials, Table S2.

#### **4. Discussion**

#### *4.1. Ponza Island Coastal front and Pelagic Biodiversity*

Biological–physical interactions structure the variability of the marine environment at a wide range of spatial and temporal scales, affecting population dynamics and trophic interactions [42,43]. Such example is a front, a physical interface with gradients of water properties that include temperature, salinity, and turbidity [44,45]. Fronts occur across the world oceans, ranging from basin-scale features to small river plumes, and can be persistent or ephemeral [44,46]. Frontal zones are often associated with enhanced biomass, and may serve as an important foraging grounds by aggregating species from multiple trophic levels [47–49], being key habitats for successful energy transfer through food webs. In situ observations and modelling studies suggest that mesoscale and sub-mesoscale processes may affect biodiversity in the Mediterranean Sea, especially where coastal morphology and intense wind stress lead to the formation of energetic filaments [50,51], contributing to the dispersal of coastal inputs towards the open sea, along with plankton.

Ponza Island is located in the western sector of the Pontine volcanic archipelago and originated during the Plio-Pleistocene on the outer margin of the Latium continental shelf. The island slope rapidly sinks to deep waters bottoms, especially on the east and south-west side (Figure 1). Winds are known to support the organic enrichment process of offshore waters, bringing the contribution of coastal freshwater input. Available time lapse records of Ponza Island and Gaeta showed three prevalent wind directions, the eastern one of which can be considered the most relevant, both in terms of intensity and frequency. This is clearly shown by the SS map presented in Supplementary Materials, Figures S1C and S2. The overlap of deep water pools, eutrophic waters, and wind driven currents may lead to an organic enrichment in the study site, due to both the upwelling and the contribution of coastal freshwater waters, supporting an increase in plankton biodiversity. Vertical transport has a well known effect in increasing the biodiversity of the photic layers only in restricted areas, as, for example, where convection is sufficiently deep, in a small number of frontal regions and in the few upwelling sites. Coastal inputs have thus an important role in sustaining food webs in the whole Mediterranean Sea [52].

Despite the data acquired in this expedition they cannot be considered exhaustive to completely depict these processes (both in terms of temporal and sampling extension), they show intriguing insights into the relation between local hydrodynamism and plankton diversity. This latter conclusion is enhanced by the record of a number of rare species, some of which appear to be far less rare than thought. An example is the case of the larval stages of the tripod fish *Bathypterois* sp., for which, to the best of our knowledge, we provide the first available photo in environment; see Supplementary Materials, Plate IV). Despite the short period of the investigation conducted in this work, over the annual cycle, mesozooplankton abundance in offshore waters of the Tyrrhenian Sea oscillated within a narrow range and revealed lower seasonal variability [53,54], with peaks occurring in April.

Moonlight has been documented to affect the distribution of plankton biodiversity, with cyclic patterns of vertical movement synchronised with variations of irradiance in all aquatic habitats [55]. Species abundances are higher during new moon and lower during full moon, suggesting that predation pressure from planktivorous species may affect plankton abundance [55]. Moon phase is also used by species to synchronize reproduction [55].

Occurrence of species during the studied period showed two different scenarios according to the moon phase. Whereas the abundance of most taxa [23] seemed not to be directly correlated only with moon cycle, for 12 taxa, there was a clear correlation between their occurrence and moon phases (Supplementary Materials, Figure S3). With the exception of Cestidae, all the other groups of moon-affected taxa clearly avoided full moon. Interestingly, field observation of some of them revealed mating behaviour, such as for Nereids (Supplementary Materials, Plate I), Sepiolidae, and Idioteidae (Supplementary Materials, Plate II), and trophic interactions (Supplementary Materials, Plate III). Juvenile and larval stages of fish were also abundant during the night with a weak moon light, as a direct consequence of lower predation rate [55] (see Supplementary Materials, Plate IV).

#### *4.2. Ecological Insight on the Uncommon Mesopelagic Fish Trachipterus trachypterus*

*Trachipterus trachypterus* is considered rare and with low population densities; however, the mesopelagic habits of the species could lead to an underestimation of its abundance [28]. This fish is only occasionally caught with professional fishing gear, and usually as one or two specimens. Most of the specimens are caught by longline or trawl at depth of 370–700 m [21,24,28,56]; in other cases, they were caught by hand, or observed near the surface or stranded [20,28]. In the present study, the species was observed and caught in exceptionally large numbers. The presence of several juveniles of *T. trachypterus* in the study area could be related to the favorable hydrological and ecological conditions typical of banks and nearby areas. Banks are important areas for fish biodiversity and often host poorly known and rare fish species [57]. Uncommon species, such as *T. trachypterus*, could be good indicators of environmental changes; however, data about the ecology and biology of this species remain scarce.

In ecological communities, it is generally believed that the principal survival option available to marine fish larvae is a fast growth and a quick enhancement of swimming performances [58–60]. The occurrence of *T. trachypterus* displayed a circa-lunar cycle, in contrast with what observed with juveniles of other fish species. A more in depth analysis showed that specimens collected referred to two different size ranges, with the bigger ones (229–157 mm) sampled during almost full moon nights (Supplementary Materials, Table S2, specimens 1 and 2) and the smallerones (91–58 mm) during

darkest nights (see also Supplementary Materials, Table S1). Despite the fact that this correlation must be supported by further observations, it is reasonable that the species behaviour changes according to the size, and that it occurs in the surface layer of the pelagic domain as a consequence of hiding or planktotrophic behaviour.

The larval fish phenotype also may have a strong impact on predation through dissembling or active defenses against predators. Diverse and complex morphologies can be found within the same family [61,62], suggesting the occurrence of different predation strategies, even if visual predation by larger fishes is likely the dominant selective force.

Batesian mimicry is a more widespread survival strategy than previously thought, especially within marine communities [63], as demonstrated by the convergence of numbers of distantly related fish taxa on the production of complex features (especially in larval stages and juveniles), like extremely long fin rays (Gadiformes, Lampridae, Lophiidae, Pleuronectiformes, and Serranidae), external guts (Gadiformes, Myctophidae), and even false eyes on stalks (Myctophidae, Stomiidae) [62,63]. Within marine pelagic environments, the evolution of visual Batesian mimicry in larval fishes must meet three fundamental conditions: (1) visual predation must be a strong source of mortality; (2) there must be a relatively common and functional model that the mimic resembles through morphology or behavior, and (3) mimics with only a slight resemblance to the model receives some degree of protection [64,65].

Within this expedition, we had the chance to observe, for the first time, some peculiar behavioural traits of juveniles of *T. trachypterus* which may be linked to Batesian mimicry.

The almost vertical position taken by juveniles of *T. trachypterus* during swimming and the dorsal–fin based locomotion recorded during our study is in agreement with the swimming behaviour previously reported for juveniles of the same species [66]. A similar swimming behaviour was recently reported also for juveniles of *T. arcticus* [67]. On the other hand, new data emerge from our study: 1- the "dorsal/ventral surface exhibition", in which the juvenile fish oriented its dorsal/ventral surface towards the observer; 2- the potential Batesian mimicry. We hypothesise that these two behaviours are related. Dorsal-orienting swimming behaviour may represent a strategy to hide the general shape of the fish and, at the same time, the extension and orientation of caudal and pelvic fins to the potential predator may enhance its appearance as a noxious (or not palatable) organism. The small occasional snap movements of these fins further recall a jellyfish species as a model for the Batesian mimicry (e.g., *Pelagia noctiluca*). Noteworthy, the elongated rays of the anterior part of the dorsal fin and the small orange expansions on these rays are similar in appearance to the cormidia of siphonophores. These peculiar morphological traits seem not to be conserved in the adult and therefore can be considered a functional adaptation to reduce predation risks during the juvenile epipelagic stage of the species. Indeed, this has already been suggested for juveniles and larvae of other mesopelagic fish species, in which particularly long and delicate fins (often in association with specific behaviours and colors) play an important role in reducing mortality due to predation [63,68–70].

#### **5. Conclusions**

Despite the numerous investigations of the last decades, the emerging picture of plankton dynamics in the Mediterranean Sea is still largely unexplored. Many factors, such as the structure of the seafloor and continental water inflows, may represent core drivers in the distribution of plankton diversity, structuring complex and dynamic trophic interactions between coastal and pelagic environments. In this research, we show how the vertical transport of coastal nutrients close to the continental shelf boundary sustains a high planktonic diversity and the occurrence of rare mesopelagic fish species. Furthermore, for a total of 12 taxa, a clear correlation between their presence and moon phases is reported.

The concept behind AQUATILIS field-based approach, along with the use of modern filming equipment, allowed us to describe, for the first time, the behaviour of the uncommon mesopelagic fish *T. trachypterus,* depicting the presence of Batesian mimicry in juveniles, and to observe and photograph rare and little known species, such as the larva of *Bathypterois* sp. Our results support the role of field studies as a fundamental instrument improving our knowledge on complex ecosystems, as those inhabited by the mesopelagic communities.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-1312/8/8/581/s1: Table S1-Planktonic taxa surveyed at the diving site; Figure S1—(a) and (b) show respectively the wind rose of Ponza (2011–2015) and Gaeta (2007-present). Map (c) and (d) show respectively the mean distribution of SST and Salinity from 1987 to present; Figure S2-In the panel (a) is presented in descending order the distribution of SST during 9th–11th of April, during 12th–18th of April and during 19th–28th of April. In the panel (b) the polar scatter of wind indicates the direction (polar orientation) and intensity of wind (x-axis). In the panel (c) is presented in descending order the distribution of Salinity during 9th–11th of April, during 12th–18th of April and during 19th–28th of April; Figure S3-Taxa occurrence according to moon phase; Figure S4. The four sampled juvenile specimens of *Trachipterus trachypterus* from the central Tyrrhenian Sea (A–C), measurements are reported in Table S2; Table S2. Morphometric measurements of the four juvenile specimens of *T. trachypterus* reported in Figure S4 and collected in the central Tyrrhenian Sea; Plate I. Mating behaviour observed during the surveyed period (A) nereids epitokes mating close to the surface; (B) details of epitokes releasing eggs; Plate II. Mating behaviour observed during the surveyed period: (A) Idioteidae on a feather; (B) Sepiolidae; Plate III. Trophic interaction within plankton community, observed during the surveyed period: (A) Scyllaridae phyllosoma feeding on a salp, with detail of its peculiar interaction with jellyfish species as a floating support; (B) *Phylliroe* cfr. *lichtensteinii* feeding on a jellyfish; (C) *Pelagia noctiluca* feeding on a salp; Plate IV. Fish juvenile/larval stages recorded during the expedition: (A) Phycidae (*Phycis* cf. *phycis*); (B) Ipnopidae (*Bathypterois* sp.); (C) Pleuronectiformes; (D) Anguilliformes. Video 1–10. Tracks of behavioural traits of interest. Available online at https://doi.org/10.5281/zenodo.3934365.

**Author Contributions:** Conceptualization, A.M., A.S., E.B. and F.T.; Methodology, A.M., A.S. and F.T.; Formal Analysis, A.M., F.P.d.M., A.D. and F.T.; Investigation, A.M., A.S. and E.B.; Resources, A.M. and A.S.; Data Curation, A.M., F.P.d.M. and A.D.; Writing—Original Draft Preparation, A.M., F.P.d.M., A.D. and F.T.; Writing—Review & Editing, A.M., A.S., F.P.d.M., A.D., E.B. and F.T.; Project Administration, A.M. and A.S.; Funding Acquisition, A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We are keen to thank the AQUATILIS teams and crew, with a special mention for Fedor Bolshakov, Dmitry Ozerov, and Pavel Kremenets. A special thanks to Sviatlana Semenova, for supporting and supervising all the field activities during the expedition. We gratefully acknowledge the AQUATILIS Association for funding the expedition and for the logistic supplies.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **The Challenge to Observe Antarctic Toothfish (***Dissostichus mawsoni***) under Fast Ice**

**Davide Di Blasi 1,2,\*, Simonepietro Canese 3,4, Erica Carlig 1, Steven J. Parker 5, Eva Pisano 1, Marino Vacchi <sup>1</sup> and Laura Ghigliotti <sup>1</sup>**


**Abstract:** In situ observation of Antarctic toothfish (*Dissostichus mawsoni*) is challenging as they typically live at depths greater than 500 m, in dark and ice-covered Antarctic waters. Searching for adequate methodologies to survey Antarctic toothfish in their habitat, we tested a miniaturized Baited Remote Underwater Video camera (BRUV), deployed through holes drilled in the sea ice in the Ross Sea region, over three field seasons. In 2015 three BRUVs were deployed at McMurdo Sound, and paired with a vertical longline sampling. In 2017, three opportunistic deployments were performed at Terra Nova Bay. In 2018 seven deployments at Terra Nova Bay provided preliminary data on the habitat preferences of the species. The design and configuration of the mini-BRUV allowed to collect high-quality video imagery of 60 Antarctic toothfish in 13 deployments from the fast sea ice. The behaviour of fish at the bait, intra-species interactions, and potential biases in individual counting were investigated, setting baselines for future studies on the abundance and distribution of Antarctic toothfish in sea-ice covered areas. This work represents the first step towards the development of protocols for non-extractive monitoring of the Antarctic toothfish in the high-Antarctica coastal shelf areas, of great value in the Ross Sea region where the largest MPA of the world has recently been established.

**Keywords:** BRUV; Ross Sea; video sampling; Antarctica

#### **1. Introduction**

The Antarctic toothfish (*Dissostichus mawsoni*) is the largest notothenioid fish inhabiting Antarctic continental waters, where it is a keystone species in the food web as a high-trophic-level predator. Since 1998, this species has been targeted by commercial fisheries in the Ross Sea (conventionally defined by the 60◦ S parallel, 150◦ E and 150◦ W meridians, and the corresponding coastline of Antarctica) managed by the Commission for the Conservation of Antarctic Marine Living Resources (CCAMLR), according to an ecosystem-based precautionary approach (www.ccamlr.org, accessed on 24 February 2021). Such an approach requires broad understanding of the species' life history and ecology as foundation for management [1]. For Antarctic toothfish, biological and ecological information has mostly been collected by observers onboard of commercial fishery vessels in offshore, deeper, and ice-free waters [2,3].

In the Ross Sea region, a large Antarctic toothfish population has been identified based on genetics and mark-recapture studies [2]. The population spans a wide geographic range from the spawning habitats in northern areas of the Pacific Antarctic Ridge to the

**Citation:** Di Blasi, D.; Canese, S.; Carlig, E.; Parker, S.J.; Pisano, E.; Vacchi, M.; Ghigliotti, L. The Challenge to Observe Antarctic Toothfish (*Dissostichus mawsoni*) under Fast Ice. *J. Mar. Sci. Eng.* **2021**, *9*, 255. https://doi.org/10.3390/ jmse9030255

Academic Editor: Francesco Tiralongo

Received: 23 January 2021 Accepted: 25 February 2021 Published: 28 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

feeding grounds on the continental slope, and juvenile habitats in the deeper zones of the continental shelf [3–5]. However, while data on size, distribution, diet and reproductive status of Antarctic toothfish from vessel-based surveys exists, it does not cover the full extent or ecological niche of the species seasonally or spatially, as vessel-based data is confined to ice free waters in summer and autumn.

Since 2018, with the implementation of the Ross Sea region Marine Protected Area (Ross Sea region MPA, CCAMLR Conservation Measure 91-05), the continental shelf has been closed to commercial fishing, further limiting information from this area, and constraining the ability to monitor the effect of the MPA. While coastal areas of the Ross Sea shelf are the main locations where toothfish are preyed upon by their main predators, Weddell seals (*Leptonychotes weddellii*) and Killer Whales (*Orcinus orca*), these fast-ice covered areas are neglected by monitoring, and few information is currently available on the Antarctic toothfish population dynamics in those areas [6–10]. Surveying through the sea ice requires large holes made through ice more than 2-m thick to take out large toothfish [8]. This is time consuming and requires the use of large and heavy equipment, both characteristics making such an extractive methodology logistically demanding in high Antarctica and limiting survey activities over large areas. To enable monitoring of Antarctic toothfish in fast-ice-covered shelf areas, innovative methods need to be developed to overcome logistical constraints, including non-extractive methods for working in the MPA.

Non-extractive methods for the study of marine fauna include acoustic and visual techniques. Underwater acoustics is largely used for studying zooplankton as well as pelagic species, but it is not effective for organisms that reside or move close to the bottom [11], such as the Antarctic toothfish, as the vertical resolution, especially at the appropriate depths, can obscure several metres of demersal habitat, and targets need to be identified from acoustic characteristics [12,13]. Conversely, underwater video techniques allow to record abundance and distribution of target species both in the water column and close to the bottom, where targets can be visually identified. Additional benefit of video sampling is the ability to observe behaviour, habitat association as well as intra- and inter-species relationships (see [11] for a review).

Among the video techniques, the Baited Remote Underwater Video systems (BRUVs) methodology is conceptually simple and based on a recording video camera that documents the arrival of organisms attracted to a baited lander [14,15]. Such a technique, which minimizes observer biases and gear selectivity associated with other survey methods, is likely appropriate for fish such as the Antarctic toothfish, characterized by good olfactory capabilities [16] and with benthic scavenger feeding habits [17]. BRUVs allow video documentation of species presence, size, and behaviour (e.g., swimming speed, feeding mode, searching). Relative abundance metrics can also be developed [18], provided that the bias associated with counting individuals that enter the field of view multiple times is accounted for [19].

Since the mid-1990s, BRUVs have been used in temperate, tropical and subtropical areas, mostly to assess the effect of Marine Protected Areas, document species behaviour, or assess changes in fish assemblages [11,20,21]. In polar waters, BRUVs have been less employed so far. BRUVs were used in the marine waters of the northern Canadian territory of Nunavut [22,23]. Very few baited camera deployments were performed in the Southern Ocean prior to present work, none in high Antarctica. An autonomous lander was deployed around South Georgia and Falkland Island to estimate the abundance of the congeneric Patagonian toothfish (*Dissosticus eleginoides*) independently from the fishery catch data [24]. A BRUV was set by SCUBA diver in shallow waters at Adelaide Island (West Antarctic Peninsula) to study the response of scavengers to feeding cues in the area [25]. Within Ryder Bay, in the West Antarctic Peninsula, a baited camera system was used to examine the distribution of scavenging fauna in relation to the spatial variation in exposure to iceberg scouring [26]. In those cases, the BRUVs were large in size and/or needed to be set underwater by a SCUBA diver, both characteristics unsuitable for work in sea-ice covered areas and at over 500 m depth.

Here, we conducted a trial of the feasibility, efficacy and reliability of a mini BRUV to study the Antarctic toothfish in fast ice-covered shelf areas. In the frame of collaborative researches between New Zealand and Italy, a pilot study was undertaken during three Antarctic field seasons, at two different locations (nearby-located to the New Zealand and the Italian Antarctic research stations that gave logistic support to make the study possible) within the Western Ross Sea region. The study aimed to (i) investigate the Antarctic toothfish behaviour at the bait and evaluate its potential influence on the calculation of relative abundance metrics, (ii) test the reliability of the results obtained by BRUV by comparison with those collected by extractive techniques, and (iii) set baselines for future studies on the distribution and abundance of the species in shelf areas.

The work promotes the diffusion of such a non-extractive technique for monitoring and sampling within the Ross Sea region MPA and sets the bases for use of BRUVs in other areas around the Antarctic continent.

#### **2. Materials and Methods**

#### *2.1. Study Area and Sampling*

The work was part of New Zealand-Italian collaborative activities conducted at Mc-Murdo Sound, near the New Zealand's Scott Base in November 2015, and Terra Nova Bay, near the Italian Mario Zucchelli Station in November 2017 and 2018.

#### 2.1.1. General Description of the Study Area

The fast-ice study area is located in the western Ross Sea (Figure 1A) with a mean depth of about 500 meters and bathymetry associated with local volcanos and scouring by glacial ice. The shape of the seafloor and depth direct tidal currents comprised Ice Shelf Water (ISW) and the water produced beneath the freezing sea ice (Ross Sea Shelf Water, RSSW). In the spring months, currents are usually less than 10 cm s−<sup>1</sup> and flow in a north-south direction [27].

All deployments were made through the 1.5–2.5 m thick fast sea ice that covers the sea surface for 9–10 months a year, from March until January. The holes extended through the platelet ice layer, a feature unique to coastal Antarctic zones [28,29]. Two locations were sampled, McMurdo Sound and Terra Nova Bay (Figure 1B). The former is a long depression up to 1000 m deep extending from the Ross Ice Shelf and bordering Ross Island [27]. The latter is along the Victoria Land coast and is a steep seafloor consisting of granitic rock ridges and gullies filled with gravel, clay, and silt [27,30]. Deployments in Terra Nova Bay were performed in Silverfish Bay which, within the vast Ross Sea region MPA, also constitutes an Antarctic Specially Protected Area (ASPA 173) of the Antarctic Treaty.

#### 2.1.2. Mini-BRUV

One of the main points for consideration when working in Antarctica is logistics. With sampling sites spanning over a large area, the constraints in the transportation, included those deriving by the use of helicopters, need to be considered. Accordingly, the BRUV system was designed to be light and portable to optimize loads during transport. Furthermore, the size of the BRUV was minimized in order to allow deployments from relatively small holes in the sea ice, thus avoiding carrying around the large and heavy equipment that would be necessary to drill large holes and saving time.

The mini-BRUV system consisted of two cylindrical housings made of Delrin® POM (DuPont™) of 70 mm external diameter and 250 mm in length. At the end of one cylinder was a 15 mm thick, flat acrylic camera and light sensor port. The other end contained a flat Delrin plate with a Seacon Electrical Wet-Mate bulkhead connector. The cylinder held a full HD Mobius camera (with a 64 Gb memory card and an Arduino Micro microcontroller board), and a NiMH battery pack. The other cylinder of the same size held a MR16 LED lamp (6 Watt, 12 Volt, cool white), and a dedicated NiMH battery pack. The two cylinders were fixed in parallel and connected by cable (Figure 2).

**Figure 1.** The study area. (**A**) Location of the study area within Antarctica continental shelf (red frame); (**B**) Sampling sites at Terra Nova Bay (left) and McMurdo Sound (right). Ice tongues or ice shelves are marked in light-blue colour; lands are marked in grey; white colour corresponds to seawater, partly covered by sea ice during the sampling periods. The sampling stations are in areas covered by fast ice in the late spring. Red dots indicate the sites sampled in 2018 at Terra Nova Bay (left) and in 2015 at McMurdo Sound (right). The green dot corresponds to the sampling site at which the BRUV was opportunistically deployed in 2017, three times with the bait and two times without the bait. (**C**) Bottom geomorphology at Silverfish Bay (Terra Nova Bay), the red dots are the seven sampling sites where Baited Remote Underwater Video camera (BRUV) deployments were carried out in 2018.

**Figure 2.** Essential structure of the mini-BRUV system. The prototype was deployed through ice holes of about 40 cm diameter.

The Arduino microcontroller was programmed to switch the camera and light "on" or "off". During the first year of field activity, at McMurdo Sound, the camera was set to perform one minute of registration and one minute of pause to optimize the battery duration. Given the good performance of the batteries in this first trial, the protocol was adjusted during the field activities at Terra Nova Bay, where registration was continuous. Approximately 1 kg of squid (*Notodarus gouldi*), routinely used in the Antarctic toothfish longline fishery, was used as the bait. It was fixed 2 m below the camera, just above the 8 kg clump weight. Three 150 mm diameter trawl floats (800 g buoyancy each) were placed above the mini-BRUV to adjust buoyancy and suspend it 2 m above the clump weight. The camera configuration was vertical with the view towards the seafloor, resulting in a field of view of about 2 m in diameter. The system was slowly lowered with a 3 mm nylon rope to the seafloor by hand or with a winch.

#### 2.1.3. Sampling Design

The study included the following three lines of investigation, covering major aspects related to the use of BRUV systems to monitor Antarctic toothfish in ice-covered areas.

#### 1. Toothfish behaviour at the bait

Three BRUV deployments were performed at McMurdo Sound (2015) and ten at Silverfish Baywithin Terra Nova Bay (three at the same station in 2017, and seven at different stations in 2018) (Figure 1B,C). In addition, in 2017, the mini-BRUV was deployed twice at Silverfish Bay without bait, as negative control to evaluate the attractive effect of the bait.

The residence time, how long on average a specimen remains in the vicinity of the bait (from first appearance to last appearance in the case of individuals that keep coming back into the field of the camera), was calculated in the 10 continuous videos performed at Terra Nova Bay during 2017 and 2018. Behaviour at the bait was evaluated as potential source of bias in the count of toothfish by BRUV [19]. When more than one individual was present in the camera field of view, the behaviour of each one was annotated and classified as "neutral", "agonistic aggressor" or "agonistic subordinate". For each agonistic event, the behaviour of both the aggressor and the subordinate fish were noted and described as follows: "stay", when the fish did not appear altered by the event; "weak reaction", when the fish reacted with rapid movements and appeared disturbed or when it escaped but returned under the field of view after a few seconds; "escape", when the fish moved away and did not return in proximity of the bait. The size of the individuals involved in the different events was included in the analysis.

#### 2. Comparison between BRUV data and longline catch

This activity was implemented at McMurdo Sound in 2015, where a scientific random stratified vertical longline survey was being conducted from the sea ice, as part of a large-scale monitoring programme [10]. The aim of this first field activity was to perform preliminary tests on the reliability of the BRUV systems in collecting Antarctic toothfish abundance data. To this end, three longline fishing stations (namely Station 20, 28, and 29, details in [10]) were considered. At each station, vertical longlines, armed with 15/0 size hooks (EZ-baiter, Mustad) baited with squid, were set three times for approximately 18 h each. The mini-BRUV was deployed at the three longline fishing stations, once per each station. The camera deployments were opportunistically performed after the retrieval of one of the longlines and before the deployment of the subsequent longline set, thus resulting in BRUV soak times ranging between 19 and 24 h. The number of individuals recorded by BRUV in a determined time of unit was compared to the vertical longline catch, expressed as average catch obtained from three replicate fishing events per station.

#### 3. Habitats and distribution of Antarctic toothfish individuals

A preliminary study, aimed at investigating the relation between physical characteristics of the sea bottom, depth and presence of Antarctic toothfish, was performed in 2018 at Terra Nova Bay. The study included BRUV deployments at seven stations in Silverfish Bay. The stations were representative of different geomorphological features (ridge and trench) and falling in a range of bottom depths from about 200 m to more than 500 m. Soak time ranged between 1 to 6 h, depending on the contingencies of the time allowed for field activity and the weather. Information on relief, substrate type, and benthos coverage of the

seafloor was recorded for each station. Depth was measured by echosounder during the video recording. The substrate type and the coverage by benthic organisms were evaluated through image analysis of the sea floor. Sediment granularity was not easily distinguishable from video footages. Therefore, we classified the substrate as "soft" when the seafloor was homogenously composed by clay, silt or sand, and "mixed" when rocky formations were visible. The benthos coverage was defined as percentage of seafloor surface in the field of view of the camera covered by epibenthic organisms. Examples of seafloor features are shown in Figure 3.

**Figure 3.** Examples of seafloor features. (**A**) Mixed substrate with high (90%) benthos coverage (Terra Nova Bay, site 05). (**B**) Soft substrate with low (10%) benthos coverage (Terra Nova Bay, site 06).

#### *2.2. Video Analysis*

Videos were screened in full with VLC Media Player 3.0.5 Vetinari Software. Identification of individual Antarctic toothfish was performed by extracting frames each time a toothfish was within the field of view and establishing individual and unambiguous key features such as colour patterns, parasites, scars, or other recognizable marks (Figure 4).

**Figure 4.** Examples of identifying marks on toothfish individuals. (**A**) Distinctive colour pattern of the pectoral fins and presence of a parasite on the left side of the trunk. (**B**) Dark spot pattern along the body.

ImageJ software was used to estimate the total length. For each individual identified, the length of the fish was calculated by comparison with the cylindrical weight on the seafloor (33 cm long) when they were both at a similar distance from the camera. In order

to minimize the effect of potential errors in the measurements, we categorised the fish length into three length classes following [3], corresponding to immature (L < 100 cm), maturing (L = 100–130 cm), and mature fish (L > 130 cm).

#### *2.3. Abundance Indices*

Relative abundance metrics, typically calculated from BRUV footages, were derived from videos recorded in 2018 at Terra Nova Bay.

The Catch Per Unit of Effort (CPUE) is an index widely used in fishery research. It was adapted to the BRUV analyses [31,32] and calculated as the total number of fish recorded, divided by the time of the video recording, and expressed as number of individual fish observed per hour. CPUE is not widely used in BRUV analysis, because unbiased value requires to identify each individual fish that enter the field of view of the camera during the entire observation period. This is especially difficult where large numbers of individuals are present, as it is often the case in shallow tropical or temperate waters, or for species whose individuals are not easily distinguishable. Given the relatively low densities of toothfish, and the possibility to identify individuals, potential bias in the use of CPUE is limited; therefore, we decided to consider this metrics.

The Mean Number (MeanN) [33], commonly used for dense and/or multispecies shoals of fish [34–44], was calculated from the maximum number of fish in a single frame from the whole video (Maximum Number, MaxN). While the advantage of this metric is to avoid recounts of same individuals [45] without the necessity to identify each individual, it can under-estimate the true abundance of fish visiting the bait, given that only a portion of the fish contribute to MaxN [46]. Therefore, we decided to consider also the MeanN, that is the average of the mean MaxN from 1-hour periods throughout the duration of the recording [33]. For the calculation of MeanN, final segments videos shorter than 1 h were discarded.

The Time of First Arrival (TFA) [47,48] was calculated as the time in minutes that passed from when the BRUV reached the seafloor and the first record of an Antarctic toothfish that entered in the field of view of the camera.

The correlation between metrics was tested through Pearson's correlation coefficient, and the relative abundance of Antarctic toothfish deriving from the various metrics was considered in the frame of a suite of environmental drivers (geomorphological features, depth, substrate type, and benthos coverage).

#### **3. Results**

#### *3.1. Fish Behaviour*

A total of 60 toothfish were observed during the 13 deployments carried out between McMurdo Sound and Terra Nova Bay. The residence time of individuals around the bait, calculated for 52 individuals during the continuous video recordings at Terra Nova Bay, was highly variable with mean residence time of 8 min 24 s ± 9 min 36 s SD (maximum 44 min, minimum 10 s). No toothfish were recorded when the BRUV was deployed without the bait. Of the observed fish, 44 were recorded close to the bait together with at least another individual in the same frame (Video S1). Most toothfish (25 individuals) were neutral to the presence of other individuals within the field of view; two individuals left the field of view without any intra-specific interaction; one was apparently involuntarily bitten by another individual and had a weak escape reaction, swimming away and returning a few seconds later; two fish increased the swimming speed in the field of view after inadvertent contact; and in 13 cases evident agonistic behaviour were recorded (Figure 5).

A descriptive analysis was developed on the 13 cases that showed agonistic events from the combination of the type of event, size classes involved, and subsequent reactions (Figure 6). In 5 cases (38.5%), the larger individuals behave as aggressors; in 5 other cases, aggressors were of the same size class of the subordinates; and 3 times (23.1%) the smaller individuals were the aggressors. The aggressors never left, and either showed weak reactions (regardless of the size of the fish attacked) in 3 cases or remained near the bait (10). The subordinates in 2 cases escaped and did not return (in both they were of the same size class of the aggressor), but in most cases they showed weak reactions and remained near the bait (Video S1).

**Figure 5.** Sequence of an agonistic event. Two toothfish are involved (left side of the images) on the side of a third individual which is eating the bait. Video recorded at Site 06, Terra Nova Bay.

**Figure 6.** Reaction of aggressors and subordinates in the 13 recorded agonistic events, according to the size of the involved individuals.

#### *3.2. BRUV Data and Longline Catch*

Sampling was performed in McMurdo Sound on the shelf area from 537 to 579 m depth (Table 1), at sites characterized by soft substrate bottom (Table S2). While BRUV was deployed for approximately 20 h at each station, in two out of the three cases, the bait was consumed prior to the end of the deployment, about one hour and after about four hours, respectively. Given the key role of the bait in this sampling system, portions of the video recorded in absence of the bait were discarded, and for the sake of standardization, the analysis of all videos was limited to the first hour of setting.

**Table 1.** McMurdo Sound, total number of fish recorded with BRUV during the first hour of setting at each station, and average vertical longline (VLL) catch values obtained from three deployments at each station.


During the first hour of the three video samplings, eight Antarctic toothfish were observed. Five (62.5%) were assigned to the immature size class, and three (37.5%) were maturing individuals.

#### *3.3. Sea Bottom Features, Depth, and Toothfish Abundance*

A total of 18 Antarctic toothfish were recorded in four out of seven BRUV samplings carried out in 2018 at Terra Nova Bay (Table S2). Most fish were in the maturing size class (*n* = 10, 55.6%), but mature (*n* = 5, 27.7%) and immature fish (*n* = 3, 16.7%) were also sighted. Eight out of ten maturing toothfish were recorded at a depth higher than 500 m (Site 06).

At sites where no toothfish were recorded, the substrate was mixed and with high percentages of benthos coverage, while in 3 out of 4 sites in which toothfish arrived to the bait, the substrate was soft and the benthos coverage low, as for McMurdo Sound. While toothfish were sighted in correspondence of ridges shallower than 400 m depth, the highest abundances were recorded in a trench habitat, at more than 500 m depth (Table 2).

**Table 2.** Environmental variables considered to characterize the 7 sites of video sampling performed in 2018 at Silverfish Bay and relative measured abundance metrics. Geomorph = geomorphology; Mean N = Mean Number; TFA = Time of First Arrival.


The values of CPUE and MeanN at the sites of sighting were strongly correlated (r = 0.95, *p* < 0.01), suggesting they may each be indicating abundance even though sample size is low. TFA did not follow the trend of the other two metrics.

#### *3.4. Other Fish Species Recorded by BRUV*

While the target species of the work was the Antarctic toothfish, other fish species, representative of the shelf demersal and pelagic assemblages, were recorded by our mini-BRUV. Juveniles and a large shoal of adult Antarctic silverfish (*Pleuragramma antarctica*) were recorded at McMurdo Sound. Juveniles Antarctic silverfish were also often recorded during the mini-BRUV deployments at Silverfish Bay. In this latter area, various *Trematomus* species were recorded. *Trematomus hansoni* were often seen approaching the bait, occasionally trying to eat it, or just swimming in the camera field in groups of five or more individuals. *Trematomus bernacchii* were also recorded around the bait, usually solitary swimming. One Artedidraconidae and one *Chionodraco* sp. incidentally entered the field of view of the camera; however, they did not show any interest for the bait. A group of *Trematomus borchgrevinki* was recorded in the upper water column, close to the sea-ice during the deployment and the hauling of the BRUV.

#### **4. Discussion**

The Antarctic toothfish is a high trophic level predator in the Antarctic ecosystems, utilizing a broad range of habitats during its lifecycle, from the epipelagic realm to benthopelagic slope habitats down to 2000 m depth [4]. Current understanding on the biology and life cycle of the species mainly relies on fishery-dependent data [2,3], resulting in catchability biases and knowledge gaps. New data on the abundance and distribution of the Antarctic toothfish in coastal shelf areas are required to support population hypotheses and management of this living resource. Large stretches of the coastal shelf areas are datapoor or unexplored, due to the occurrence of fast sea ice that prevents the access of fishing vessels. This holds truth even in regions where the toothfish is historically harvested, such as the Ross Sea region.

We investigated the performance of a BRUV system to collect biological data on the Antarctic toothfish in the data-poor sea-ice covered areas of the Ross Sea region shelf, a region of interest for fishery management and marine conservation.

BRUV systems were demonstrated to produce results comparable to some fishery-based methods for monitoring trends in the relative abundance [31] and have been proven effective in surveying mobile predators and/or opportunistic scavengers, such as sharks [22,37] and grenadier [49], thus making this sampling technique promising to collect fishery independent data on the predator and scavenger Antarctic toothfish [50]. Prior to the present work, a few attempts were made for the use of BRUV systems in the Antarctic waters. In the West Antarctic Peninsula, a BRUV was used to examine scavenging fauna in relation to the exposure to iceberg scouring within Ryder Bay [26], and a preliminary study was performed on the use of baited cameras mounted on a rather large autonomous lander (Aberdeen University Deep Ocean Submersible (AUDOS)) to estimate the abundance and size of the co-generic Patagonian toothfish *D. eleginoides* in sub-Antarctic areas [24]. In both cases, the activities were in open waters, with rather voluminous baited camera systems deployed from boats and set on the sea bottom.

The study on *D. eleginoides*, while demonstrating the general feasibility of toothfish abundance estimation by BRUV, stressed the need for improvements in the design of the system and suggested the use of short-term deployment times (2–3 h). Following those recommendations, in the present study, the efforts were put in the design and configuration of the system, as well as in the optimization of the protocol.

In line with recent trends towards the development of miniaturized deep-sea cameras targeting the reduction of size and costs [51], our BRUV system was developed to be essential in the design and small in size and set up with the camera view facing vertically downward. The majority of BRUVS set-ups used a horizontal camera arrangement, and only the 14% had a vertical orientation pointing down to the seafloor [21]. The vertical camera setting is underused because some species seem reluctant in entering the vertical field of view, most likely due to the perceived confined space under the camera, emphasized by the occurrence of large aluminium frames. Our essential BRUV system does not present any frames or bait arms (the bait is set close to the weight connected by a short piece of fishing line), while camera and light are suspended at about 2 m from the seafloor. Such a setting did not seem to affect the fish behaviour, and Antarctic toothfish were recorded swimming around the bait for quite long time, about 8 min on average, and up to 44 min. In this configuration, the mini-BRUV system was proven suitable for deployment through holes in the sea ice of relatively small diameter (even less than 40 cm), significantly reducing the workload and logistics required to perform surveys from the sea ice. Furthermore, it was easily transportable and light enough to be set and hauled by hand.

In order to optimize the protocol, soak time was carefully considered. During the field work at McMurdo Sound, the association of BRUV work to the longline activities imposed long duration of deployments, with soak times of over 20 h. However, the a posteriori analysis of the videos clearly demonstrated that long deployments are unnecessary and that soak time from one to six hours is ideal, allowing both data collection and multiple deployments in a short period. Remarkably, no decay in the effect of the bait was recorded in such a timeframe, and Antarctic toothfish were spotted approaching the bait until hour six. Overall, the effectiveness of short-term deployments to monitor *Dissostichus* species [24] is confirmed; one-hour video record is likely sufficient to collect baseline data; however, deployments up to six hours might allow for a more precise calculation of relative abundance metrics.

A critical point for the optimization of protocols for BRUV observations is the effectiveness of the bait in attracting the target species, which has repercussions on abundance metrics [39,52,53]. Here, the bait was a squid routinely used in the Antarctic toothfish longline fishery. The behaviour of the fish at the bait supports a positive olfactory response and searching behaviour in the odour plume, which is expected based on their olfactory capabilities [16]. The fish were observed approaching slowly to the bait, often sliding it along their flank and eventually grasping it in their jaws. *Dissostichus eleginoides* individuals were reported to be attracted by the bait but never observed to investigate closely or attempt to take the bait [24]. Such distinct behaviours could be related to differences in

the BRUV configurations. In particular, the essential design of our mini-BRUV was likely perceived by fish as not disturbing and did not arise suspicion on fish. However, it is also worth noting that the work on *D. eleginoides* was performed in a commercially longlined, plenty-of-food area, while our activity on *D. mawsoni* was performed in areas not accessible to fishing vessels and, in the case of Terra Nova Bay, in the frame of a specially protected area (ASPA 173) where disturbance of anthropic origin was minimal.

The effectiveness of the bait in attracting Antarctic toothfish individuals was also supported by the absence of any fish record in the negative controls held with no bait. The effect of other potential attractants related to the BRUV, such as light and noise, or presence of other organisms around the system, seems negligible. Interestingly, while the use of intense light in BRUV is usually discouraged due to possible flash induced bait shyness [24], the Antarctic toothfish individuals observed under the light of our mini-BRUV did not show any discomfort; on the contrary, some of them were attracted by the light and occasionally were recorded swimming upward pointing the lamp (Video S1).

The design and configuration of our mini-BRUV allowed to collect high-quality video imagery of 60 Antarctic toothfish in 13 deployments. To the best of our knowledge, only brief images of a single Antarctic toothfish, incidentally acquired during a video survey in McMurdo Sound [54], and sparse snapshots of fish from cameras attached to Weddell seals [55] were available prior to this work. Here, the high quality of the videos is coupled with high number of records, thus providing valuable new documentation of the Antarctic toothfish in its natural habitat (Video S1). Owing to the quality of the videos and the low number of individuals occurring in the field of view simultaneously, identification was possible for all the individuals. Scars and unique colour patterns present were effective natural markings to distinguish individuals and, although the persistence over time of those markings is unknown, at least they can be used as effective markers within a single deployment. This facilitated the abundance metric calculations, and biases related to re-counting were avoided.

Two of the three used metrics, CPUE and MeanN, resulted strongly correlated, but their efficacy remains to be confirmed with additional data. The possibility to use MeanN as a proper abundance metric would allow to avoid the identification of single individuals, a difficult and time-consuming step necessary for the calculation of CPUE, thus significantly reducing the time allocated to video processing, which is necessary. This would foster the application of the methodology and the collection of a large number of recordings. Among the other metrics considered, the time of first arrival (TFA) was successfully adopted for the congeneric Patagonian toothfish [24]. Such a metric is powerful when integrated with data on the current and fish swimming speed [51]. However, in the present study, due to the lack of current speed measurements and reliable estimate of the speed of fish attracted by the bait, the TFA could not be standardized, and its informative value as proxy of Antarctic toothfish abundance was poor.

The low number of BRUV deployments for comparison with longline catch in Mc-Murdo Sound prevented any robust calculation of metrics or trends. However, at the stations where the catch with longlines was the highest, Antarctic toothfish individuals appeared in the field of view of the camera during the first hour, supporting the relation between fish catches and BRUV counts. A relation between the calculated BRUV abundance metrics and the longline catch rates seems to be occurring, but further comparative investigations are needed to provide statistical support to this observation.

Besides quantifying the relative abundance and distribution of target species, the BRUV systems hold potential to generate a variety of data; characterize benthic habitats; and assess functional diversity, body sizes, and animal behaviours [22], thus, in turn, facilitating investigation of fish–habitat relationship [56]. We collected basic information on the sea bottom features (composition and granulometry) from all videos and conducted a preliminary study to investigate the relationship between physical characteristics of the environment and presence of Antarctic toothfish at Terra Nova Bay. Regardless of depth and geomorphology, toothfish were recorded at three stations characterized by soft bottom

and relatively low benthos coverage and only one station with a mixed sea bottom. The preference toward soft sea bottom seems confirmed by the data from McMurdo Sound, where toothfish were BRUV sampled only in soft bottom areas.

Another interesting observation from the videos is the size of the Antarctic toothfish at Terra Nova Bay, larger than that expected for the Ross Sea shelf area [3], with prevalence of individuals between 100 and 130 cm length (55.6%) and some larger mature ones (27.7%). It is worth noting that the fish sampled by BRUV at McMurdo Sound are overall smaller than the ones observed at Terra Nova Bay. Despite being a point observation, this aspect might have repercussions on current population hypothesis for the Ross Sea and deserves further investigation.

Overall, the results of our trials, encourage the use of BRUV to study the abundance and distribution of Antarctic toothfish in sea-ice covered areas but also as a valid investigation tool for field work in other areas around the Antarctic continent, whether or not seasonally covered with fast ice. Here, clues for the optimization of the sampling protocols, including information on the bait and optimal soak time, are provided. Furthermore, this study, although preliminary, identified gaps in the knowledge base that could be addressed by the use of BRUV, including habitat preferences of the Antarctic toothfish and size distribution in ice-covered shelf areas. As a side information, our mini-BRUV was proven effective in attracting fish species other than our target species and could thus be used to study densities and behaviour of those species, as well as fish assemblages.

Our pilot investigation allowed to highlight that some improvements should be considered in future studies. In particular, the addition of a current meter would allow to consider current speed that might influence the odour dispersal and lead to bias in the metrics calculations, especially TFA [24]. Another important improvement would be the addition of coupled lasers pointer with parallel light beams, which would allow a more accurate calculation of the fish length and evaluation of the size distribution for the species. Furthermore, the laser pointers would aid in estimating the fish swimming speed, another key element that influences the TFA and, consequently, the abundance estimate. The setting up on the BRUV of sensors for water parameters might add relevant environmental information in support of habitat preferences evaluations. The description of water mass preferences of adult toothfish might provide relevant information on the movements of adults on the continental shelf, with repercussion on the management of the species. The use of non-extractive methods, including BRUV, is of particular relevance in the Ross Sea region, where the largest MPA of the world has recently been established and where this tool can be used in many more applications to study target species but also assemblages and behaviour, supporting research and monitoring in the area.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2077-131 2/9/3/255/s1. Video S1: Behaviour of Antarctic toothfish (video recorded on November 14, 2018, at Site 06, Terra Nova Bay). Table S2: Summary table.

**Author Contributions:** Conceptualization, L.G. and M.V.; methodology, D.D.B. and E.C.; validation, E.P.; formal analysis, D.D.B.; investigation, D.D.B., E.C., S.C., and S.J.P.; resources, S.C.; data curation, D.D.B.; writing—original draft preparation, D.D.B.; writing—review and editing, E.P., L.G., and S.J.P.; supervision, L.G., S.J.P., and M.V.; project administration, L.G.; funding acquisition, L.G., M.V., and S.J.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Italian National Programme for Antarctic Research (projects MIUR-PNRA 2015/B1.02 – DISMAS, MIUR-PNRA 2016/AZ1.19 – PILOT) and by the New Zealand Ministry for Primary Industries (project ANT201501) and Antarctica New Zealand.

**Institutional Review Board Statement:** In situ activities were carried out at McMurdo Sound in accordance with permit AMLR15/R01/Parker/K086 issued by the New Zealand government under the Antarctic Marine Living Resources (AMLR) Act 1981 and at Terra Nova Bay in compliance with the Protocol of Environmental Protection to the Antarctic Treaty, Annex II, Art.3, in the frame of PNRA Research Projects.

**Data Availability Statement:** The data presented in this study are available in article and in the Supplementary Materials.

**Acknowledgments:** We thank the CCAMLR EMM and FSA Working Groups participants, with whom the work was discussed, for fruitful suggestions on the refinement of the technique. The participation of Davide Di Blasi in the CCAMLR Working Groups was allowed by the CCAMLR Scholarship for early career researchers during years 2018–2019. Part of the work was carried out during his PhD at the University of Genoa. We thank the three anonymous reviewers for their thoughtful comments and constructive remarks.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**


#### *Article*
