Population structure of the invasive Atlantic blue crab, Callinectes sapidus on the Eastern Adriatic coast (Croatia, Montenegro) Struktura populacije atlantskog plavog raka, Callinectes sapidus na istočnoj obali Jadrana (Hrvatska, Crna Gora)

* The Atlantic blue crab, Callinectes sapidus , is a highly invasive species that poses a signifi cant threat to Mediterranean ecosystems. In the last two decades, it has become established in several marine and estuarine areas of the eastern Adriatic Sea, resulting in a decline in commercial catches and damage to fi shing gear. This article reviews the current status of blue crab invasion in Montenegro and Croatia and analyses its abundance and population structure. Overall 619 crabs were sampled (male:female ratio, 1:1.91). Both carapace width and weight diff ered signifi cantly between males and females, with males having a wider carapace and more weight. There was a signifi cant diff erence in carapace width and weight among sites. For the total population, the mean male and female carapace width was 130.3 ± 30.8 and 108.8 ± 41.4 mm, respectively. In addition, the mean male and female weight was 187.2 ± 85.6 and 132.5 ± 39.1 g, respectively. Coeffi cient b between the weight and carapace width of blue crabs was signifi cant at all locations, although it varied between males and females. This work will also document the impacts of the blue crab invasion on local ecosystems and provide comprehensive overview of populations structures and shed light on this important aspect of blue crab ecology.

The Atlantic blue crab, Callinectes sapidus, is a highly invasive species that poses a signifi cant threat to Mediterranean ecosystems.In the last two decades, it has become established in several marine and estuarine areas of the eastern Adriatic Sea, resulting in a decline in commercial catches and damage to fi shing gear.This article reviews the current status of blue crab invasion in Montenegro and Croatia and analyses its abundance and population structure.Overall 619 crabs were sampled (male:female ratio, 1:1.91).Both carapace width and weight diff ered signifi cantly between males and females, with males having a wider carapace and more weight.There was a signifi cant diff erence in carapace width and weight among sites.For the total population, the mean male and female carapace width was 130.3 ± 30.8 and 108.8 ± 41.4 mm, respectively.In addition, the mean male and female weight was 187.2 ± 85.6 and 132.5 ± 39.1 g, respectively.Coeffi cient b between the weight and carapace width of blue crabs was signifi cant at all locations, although it varied between males and females.This work will also document the impacts of the blue crab invasion on local ecosystems and provide comprehensive overview of populations structures and shed light on this important aspect of blue crab ecology.

INTRODUCTION
The Atlantic blue crab, Callinectes sapidus, Rathbun, 1896, is a portunid brachyuran native to the western Atlantic Ocean from Nova Scotia to Uruguay and Argentina [1].The fi rst appearance of the blue crab in European waters was reported in 1901 in Rochefort, France [2], where it was probably introduced with ballast water [3].Since then, the species has spread to the Mediterranean Sea, where it was fi rst described in 1947 and has spread widely in the northwestern areas of the Mediterranean, including the Adriatic Sea [4,5].The blue crab's excellent swimming abilities, opportunistic feeding behaviour, broad habitat preferences, high fecundity, and wide larval distribution are all biological characteristics that contribute to its invasive nature and allow it to spread successfully in diff erent regions [6].These characteristics have led to its recognition as one of the 100 worst invasive alien species in the Mediterranean [7].The fi rst detection in the eastern Adriatic was in 2004 in the Neretva estuary and in the salt pans of Ston [8], and a strong population was established in the Neretva estuary after six years [9].The fi rst information about the occurrence of blue crab in Montenegro, specifi cally in Port Milena, comes from a personal observation in 2006 [10].The fi rst verifi ed record dates back to 2013, when two males were caught with a gill net in Boka Kotorska bay, while the fi rst female was discovered in 2017 in the lagoon of the Tivat Solila nature reserve [11].This study addresses the population characteristics of blue crab using common tools such as morphometrics, length to weight ratio and sex ratio.In addition, the study provides quantitative data on allometric growth and qualitative data on the biology and ecological preferences of the blue crab in its non-native range in the eastern Adriatic Sea.Samples from Montenegro and Croatia were analysed to provide new insights into population structure that will be valuable to scientists, policy makers, and those involved in the management of non-native and invasive species, and will contribute to the understanding and management of the blue crab population in the aff ected regions.

Sampling area
Samples of blue crab were collected between 2018 and 2021 at six diff erent sites, three of them in Croatia and the remaining three in Montenegro.The study area in Croatia was located in Dubrovnik-Neretva County and included the rivers Neretva (Parila lagoon), Ston and Mlini, which diff er signifi cantly in terms of water temperature and salinity.The Neretva and Mlini areas are characterized by the infl ow of fresh water into the sea, but in diff erent ways.The Neretva River (43°01'55.3"N 17°27'05.2"E) has a main channel with lagoons on both sides, which are not aff ected by strong river currents and have a higher temperature and salinity than the surrounding areas.The temperature in the delta of the Neretva River ranges from 9.2°C to 26.7°C and the salinity from 8.0 to 34.4 ppt (in the Parila lagoon from 7.2°C to 33.4°C and the salinity from 11.4 to 34.6 psu.Mlini (42°37'18.0"N 18°12'19.0"E), on the other hand, is a small town that owes its name to the old mills still located in the old part of the town.These mills were located on the freshwater rivers of the area, and in 1952 a small hydroelectric power plant with a generator called "Zavrelje" was built, using water from the same river.The water is discharged to the sea through an outfl ow channel, and to reduce the impact of sea waves on the Zavrelje, a breakwater was built.The site in Ston (42°49'44.4"N 17°41'53.4"E) is characterized by shallow water with low circulation, walled with stones, which serves as a basin for the company Saline Ston.The term "saline" refers to a facility or site where salt is produced by the evaporation of salt water.The water for the salt works is transported to the basins through a series of canals that remain unused for most of the year.As part of the study, these canals and the adjacent lagoon were used as sampling sites to monitor the presence of blue crab.
The Bojana River (41°52'30.0"N 19°22'24.3"E), together with the Port Milena wetland and Šasko Lake, is the largest body of water on the entire Montenegrin coast [12].The Bojana River is 43 km long, starting at Lake Skadar and fl owing through Albania for 18 km before marking the border between Montenegro and Albania for the remaining 25 km.At its mouth into the Adriatic Sea, the Bojana River forms a small delta with two side channels, the left of which forms the border with Albania and the right of which, together with the island between the channels, belongs to Montenegro.Island of Ada Bojana today represents a unique ecosystem in Europe [13] and covers an area of 6 km2.The water temperature in Bojana varies between 4.8°C and 25°C throughout the year.Salinity ranges from 0.1 to 2.6 psu, but can reach up to 20 psu in the lower water layers near the delta during strong southerly winds.Port Milena Channel (41°54'56.3"N 19°15'11.8"E) has been transformed from a natural lagoon into a channel whose cleaning is obstructed by the Bojana River, dammed by a salt processing plant built in 1934.Hladna Uvala (41°59'38.4"N 19°08'41.4"E) is a fi shing cove between towns Bar and Ulcinj, where there are several freshwater springs.

Sampling and gear description
Sampling was conducted with two types of fi shing gear.The fi rst was a locally remodelled version of American wire crab traps with a rectangular shape, 60 cm long, 60 cm wide, and 40 cm high, a mesh size of 40 mm, two entrances, and a compartment for storing bait (Figure 2A).The second type of gear used in the study was gillnets.Gillnets are vertical fi shing nets suspended in the water and anchored to the bottom.The net material consists of a mesh large enough for the fi sh to fi t their head through, but not their body.The fi sh's gills then become entangled in the mesh when the fi sh tries to free itself from the net, hence the name "gill net." Fish waste from non-commercial or low-value species such as thin-lipped mullet (Chelon ramada) was used as bait for the wire traps.In addition, Gibel carp (Carassius gibelio) and pumpkinseed (Lepomis gibbosus) were also used as bait for the traps [14].Both types of gear were deployed in the afternoon.The traps were soaked for 24 hours while the gillnets were hauled in the next morning.Catches from the crab traps were collected (Figure 2B), the traps were rebaited and returned to the sea, while the gillnets were brought ashore and cleaned manually.
After collection, specimens were identified to species level based on Galil et al. (2002).Weight (W) and carapace width (CW, maximum distance between posterior anterolateral spines) and sex were measured for all 619 individuals.Measurements were made using a digital caliper (Mitutoyo) for carapace width and a digital scale for weight (Mettler Toledo JL602-G/L).2.3.

Statistical analysis
Statistical analyses were performed using SPSS Statistics v.26.Mean diff erences in carapace width (CW) and total weight (W) between males and females were tested with an independent T-test.Diff erences in sex ratio between sites were tested with the nonparametric chi-square (χ2) test.Mean diff erences in carapace width (CW) and total weight (W) between sites were tested using the 1-way test ANOVA.If a statistically signifi cant diff erence was found, a Tukey HSD post hoc test was performed for multiple comparisons.Diff erences in b coeffi cients between males and females were tested with an independent T test and between sites with a 1-way test ANOVA.Before analysis, data were visually checked for normality with Q-Q plots and tested for homogeneity of variance with Levene's test.All assumptions were met.For all tests, the signifi cance level was set at 0.05 (α = 0.05).

Distribution
A total of 619 crabs were sampled (212 males and 407 females), (male:female ratio 1:1.91).Sex ratios diff ered signifi cantly among sites (χ2 (5) = 33.49270,p = .001);ratios at each site are shown in Figure 3.In Montenegro, in the Ada Bojana site the most crabs were captured, 149 females and 48 males (197 total).In Croatia, the most crabs were caught in the Neretva River delta, including 118 males and 44 females (162 total) (Table 1).
For the total population, the mean carapace width (CW) of males was 130.3 ± 30.8 mm with maximum and minimum values of 182 mm and 51.5 mm, respectively.The mean carapace width of females was 108.8 ± 41.4 mm with a maximum and minimum value of 182 mm and 45.1 mm, respectively.In addition, the mean weight (W) of males was 187.2 ± 85.6 g with a maximum and minimum value of 449.2 g and 17.2 g, respectively.The mean weight of females was 132.5 ± 39.1 g, with maximum and minimum values of 256 g and 13.7 g.A summary of data for the Croatian and Montenegrin catches at each site is presented, including mean carapace width (CW) and weight for males and females (Table 1).

Length-weight relationship and length-weight population structure
The relationship between carapace width and weight was calculated for both sexes of blue crab using power regression: W=a×Lb, where W is weight in grams, L is carapace width in centimeters, a is the intercept of the regression, and b is the slope [15].A strong correlation was found between the weight and carapace width of crabs, as shown by the high correlation coeffi cients for the sites in both countries (Figure 4).However, the slope b for males and females varied considerably between sites, with the lowest value being 1.1125 for Hladna Uvala (where only four males were captured) and the highest being 3.1607 for males from Port Milena (Table 1).Blue crabs were divided into carapace width and weight classes and presented in diagrams (Figure 5).The greatest abundance of males is found in the 140-149 mm CW class and females in the 60-69 mm CW class.The majority of males fell into the 130-149 g weight class, while the majority of females fell into the 110-129 g weight class (Figure 5).There was no signifi cant diff erence in b coeffi cients between males and females (t(10) = .144,p = .889)or between sites (F(5,11)= 2.666, p = .132).

DISCUSSION
The study compared populations of blue crabs sampled at diff erent locations along the southern east coast of the Adriatic Sea.The highest number of crabs was recorded at the mouth of the Bojana River (Montenegro), with a predominance of females in the total catch, while samples from the Neretva River estuary (Croatia) contained dominantly males.It should also be taken into account that in the area of Ston saltworks a majority of males were caught, and in Port Milena in Montenegro a majority of female crabs were caught, since in both cases they are lagoons located near saltworks with similar water conditions, but with opposite sex distribution.This indicates that sites with similar ecological conditions do not necessarily have similar population distributions.The village of Mlini in Croatia, closest to the border with Montenegro, is the only site with a similar sex distribution to all three sites in Montenegro.The similarity of catch data in Mlini and Hladna Uvala, both small bays with freshwater springs, suggests that females on the eastern Adriatic coast prefer marine areas with colder water in summer, as males were almost absent at these sites.This result is consistent with previous observations that males are more abundant in lower salinity environments as females migrate to higher salinity coastal waters to spawn [16].This study has shown that the population structure of this species in these areas is characterized by a high proportion of females, a size distribution toward larger male individuals, and a high reproductive potential.In addition, as a strong swimmer, the blue crab can migrate between the sea and rivers throughout its life cycle, resulting in strong geographic dispersal and colonization [3].Nevertheless, blue crab is an important target for commercial and recreational fi sheries in its native range [17].In the U.S., the commercial fi shery for this species is estimated to be worth between $141 million and $205 million between 2000 and 2019, according to the National Marine Fisheries Service [18].Because most stocks of commercially valuable crustacean species are currently in critical condition, new fi shing grounds and species should be developed [19].In some parts of Europe, such as Greece, this species has become a target of commercial fi sheries [2].It is expected that the future development of bottom and coastal fi sheries in the Mediterranean will follow the changes already observed at the European level [19].Creating a sustainable blue crab fi shery that can transform invasive populations from a threat to an opportunity for valuable fi shery resources with real market value and demand is crucial management step in control of this species.Involving tourists in the fi shery and promoting blue crab as food could help regulate the crab population, which is important since both countries rely heavily on tourism.

Figure 1
Figure 1 Sampling locations of the Atlantic blue crab Callinectes sapidus (Rathbun, 1869) in Croatia and Montenegro

Figure 3
Figure 3 Sex ratio of the Atlantic blue crab Callinectes sapidus (Rathbun, 1869) at each sampling site on the Eastern Adriatic Sea coast

Table 1 A
summary of catch data for the Atlantic blue crab Callinectes sapidus (Rathbun, 1869) at each site (CW = carapace width)