Diet of the blue crab (Callinectes sapidus) during range expansion in Great Bay Estuary, New Hampshire

Abstract

Great Bay Estuary (GBE), within the rapidly warming Gulf of Maine, has experienced significant ecological shifts this century due to naturalization of invasive species. The range expansion of the American blue crab (Callinectes sapidus) currently underway from the mid-Atlantic northward brings the possibility of similar ecological shifts. This study accounts recent trapping and diet analysis of C. sapidus in GBE. Diet is an important component of understanding how the blue crab range expansion may affect GBE ecosystem functions. Across all sites and trap types, 27 blue crabs were captured. Metagenomic analysis of shotgun sequencing techniques were used on the gut contents of blue crabs captured. Most specimens had > 50% Eukaryote sequences. Overall results of this gut content study confirm a mixed diet indicative of an opportunistic feeder. Using metagenomics to analyze the diet of blue crabs as they establish viable populations in GBE will be a useful tool for predicting how these range expanding organisms are interacting within this important estuarine ecosystem, which will promote sustainable development by informing end users who may be affected by these crabs to help them meet their needs in the present and future. This project falls within Global Goal SDG14: Life Below Water.

Background

For the past century, the Atlantic blue crab, Callinectes sapidus [3], has been expanding its range and invading new areas [4,5,6,7]. Concomitant with climate change, species distribution [1, 2] is changing through increases in water temperature and via unintentional introductions. Although species distribution through both climate driven site suitability and human mediated transport interact, they also can be separate paths for expansion. For example, with water temperatures increasing, blue crabs may move as new areas become suitable habitats. This type of climate driven range expansion of blue crabs has been on notice in the Gulf of Maine [4]. The native range of blue crabs extends along the Atlantic Coast of the Americas including Gulf of Mexico [9], and southward to South America [8, 9, 63], with expatriates noted in Maine [10], and northward to Nova Scotia [4, 7, 11]. Blue crab range expansion in the Gulf of Maine has been studied [4] and recently, mated pairs of blue crabs were observed in Great Bay Estuary, New Hampshire [11].

Great Bay Estuary (GBE) is part of the Gulf of Maine region, which is experiencing some the fastest rates of climate change [12,13,14], resulting in increased sea surface temperature [13]. Warmer waters due to climate change can alter marine organism distributions [15], feeding activity [16], interspecific interactions [17], effectiveness of species invasions [18], and range expansion [19]. As this range expanding blue crab, a possible predator of benthic species and prey of pelagic fishes, finds a more hospitable habitat, it is possible that a population of sustained numbers will ensue in GBE.

The arrival of non-native species to New England, specifically GBE, is not a new phenomenon. For example, invasive species such as Botrylloides sp. (tunicate) [20], Carcinus maenas (European green crab) [20,21,22,23], and Hemigrapsus sanguineus (Asian shore crab) [20], now are naturalized in GBE, with documented effects [21, 24] and predicted effects [20]. Assuming the blue crab population continues expanding in this unique estuary, there is much interest in its potential impact upon existing ecologically important keystone species in the bay such as oysters [25, 26, & 27] and eelgrass [28, 29, 30, & 31], which could be negatively impacted by blue crabs if they substantially alter the GBE food web. This study provides the first published data for the numbers of blue crabs in GBE, obtained through both bycatch and intentional capture in 2022 and 2023. A snapshot of the diet of those blue crabs was performed using metagenomic analysis of their gut contents [32].

Materials and methods

Blue crab sampling

GBE is a well-mixed estuary located on the southern New Hampshire-Maine border [33]. Beginning in 2020, a trapping system targeting the European green crab (Carcinus maenas) was instituted, but only in 2022 and 2023 were blue crabs (Callinectes sapidus) captured; therefore, summary of methods and data only for those two years are presented here. In 2022, four sites throughout the estuary were selected for sampling using trapezoidal green crab traps (n = 3 per site) from Brooks Trap Mill, Maine. The traps measured 0.91 m L × 0.46 m W × 0.28 m H and were made of 4 × 4 cm coated wire mesh. Nannie Island (NI) and Moody Point (right outside Lamprey River area) (MP) were located near known historical native oyster reefs, and Cedar Point (CP) and Fox Point (FP) were located near known oyster farms. These four sites cover the upper and lower bay areas of GBE (Fig. 1) [34].

Fig. 1

Map of GBE showing four sites (blue diamonds) where trapezoidal crab traps and crab pots were deployed: CP, FP, NI, and MP, and areas where two crab pots (orange diamonds) were deployed: BKC and OR. For reference, SR: Squamscott River, LR: Lamprey River, OR: Oyster River, PR: Piscataqua River, and WR: Winnicut River are five of the seven rivers that flow into Great Bay

During April-November 2022, green crab traps were baited once per week with herring or mackerel placed inside stainless-steel tea bags that lured crabs into the traps but prevented them from consuming the bait, so that they would not skew diet results, and checked twice per week. Although traps were baited (but bait was not available for consumption) and may attract other species along with blue crabs, these data provide an indication of what they would consume in this environment. In collaboration with New Hampshire Sea Grant, starting in July, blue crab pots (0.60 m L × 0.60 m W × 0.5 m H, 4 cm hex mesh) were deployed alongside each n = 3 trapezoidal trap, also with bait sequestered in tea bags, with the intent to capture blue crabs. All traps were checked at high tide, 48 h after baiting to ensure that the trapped crabs could have had full or semi-full gut contents. This was based on the findings of prior studies that found crabs starved for three days had empty guts [35]; however some studies have shown the blue crab gut system is emptied in 18–24 h [56], meaning most gut contents of blue crabs captured likely were intact when the crabs entered the traps or else were from items consumed during their time in the traps. Blue crab bycatch became a frequent occurrence starting in September and continuing through October (Figs. 2 and 3) in these green crab trapezoidal traps. In 2023, the trapping was repeated with the full intention of catching blue crabs, but with one typical blue crab pot alongside one trapezoidal green crab and one rectangular trap (0.61 m L x 0.23 m W x 0.46 m H) at all sites, checked weekly from April-November. As the previous year, blue crabs again were captured in late summer until early autumn.

Blue crabs also were captured in an Oyster River subtidal zone by NH Fish and Game (NHF&G) in the process of their annual fish seine sampling, and by Great Bay National Estuarine Research Reserve (GBNERR) personnel using blue crab pots deployed in Bunker Creek, intertidal and salt marsh areas. All blue crabs captured were sexed, weighed, and measured (Table 1), and when guts were present (i.e., crabs with rigid exoskeletons; soft crabs that recently completed ecdysis had no gut contents) the gut contents were analyzed for diet using whole-genome shotgun sequencing of DNA extracted from the gut contents. Metadata including latitude and longitude of sites, dates of blue crab sampling, mass and carapace width of blue crabs, and habitat characteristics were compiled into a single table (Table 1).

Fig. 2

Trapezoidal green crab trap with both blue crabs and green crabs. Nannie Island, 21 September 2022

Fig. 3

Male blue crab caught on 19 October 2022 in a trapezoidal green crab trap at Moody Point-Lamprey River area

Table 1 Metadata for mature blue crabs and sampling areas where they were captured

DNA extraction, sequence acquisition, and statistical analyses

Blue crabs were frozen on the day of capture and subsequently thawed. The gut of each blue crab was excised, and gut contents were pressed into a microfuge tube, about 200-250µl. DNA from blue crab gut samples (all hard carapace adults) was extracted using a QIAGEN DNAeasy PowerSoil Kit^® according to the manufacturer’s instructions and total DNA was evaluated for quantity/quality using Qubit 4 Fluorometer (Thermo Fisher Scientific) using Qubit 1X dsDNA HS Assay kit (Invitrogen). Sequencing libraries were prepared using the Kapa BioSystems HyperPlus Kit (KR1145 -v3.16). Sequencing was completed on a NovaSeq 6000 with an SP flow cell (paired-end 250 bp reads). Data were demultiplexed using bcl2fastq v2.20.0.422 after which FASTQ files were examined for quality using FastQC v0.11.5 and multiqc v1.11 with default settings [36]. Estimation of duplication rates, adapter sequence trimming, and read merging was completed using fastp v0.23.2 with [37] default settings and the flags ‘--include_unmerged’, ‘--merge’, ‘-l 50’ and ‘-g’ to remove poly-G tails produced by the NovaSeq sequencer.

Translated reads were aligned to the NCBI nr database using the DIAMOND v2.1.9 [38] with BLASTX command with default settings and the ‘-f’ option to write a diamond database file (DAA). Taxonomic and functional classifications were completed using the daa-meganizer tool, implemented in the MEGAN (MetaGenome Analyzer) v6.25.9 software package [39], (see python script in Supplemental Material). Taxonomic classification was performed using the NCBI taxonomy and the GTDB taxonomy [40] and the naïve Lowest Common Ancestor (LCA) algorithm [41]. Classification was enabled with a minimum score threshold of 50.0, a maximum expected value of 0.01, and a top percentage of 10.0 considered for reporting resulting reads being assigned to taxonomic classifications based on read count, using a naive LCA algorithm with 100.0% coverage and a minimum support threshold of 0.01. Relative abundance normalization [42] and construction of a BIOM formatted table was completed using a custom python script. Data were aggregated to the taxonomic rank of Genus and statistical analyses were performed at the Family level. The method we selected for taxonomic classification is robust. The LCA algorithm has been widely adopted and validated in the scientific community, demonstrating its reliability and robustness across various studies and applications. The LCA algorithm assigns taxonomic classifications by tracing the hits of each read through a taxonomic hierarchy. This allows it to place sequences at the most specific taxonomic level supported by the data, providing a balance between specificity and accuracy. By considering all possible taxonomic assignments and choosing the most common ancestor, the LCA algorithm minimizes errors that can arise from ambiguous or conflicting hits. This conservative approach reduces the likelihood of false positives. In addition, the LCA algorithm is resilient to incomplete or imperfect databases. By assigning sequences to the lowest common ancestor, it avoids over-specification, which is particularly useful when reference databases do not contain exact matches or are hindered with mis-identified sequences. Sequence data are deposited in the NCBI SRA under the BioProject accession PRJNA1090924.

Normalized taxonomic arrays were analyzed using Several-samples Repeated Measures Tests (ANOVA) [43], Tukey’s studentized range test [44], and Wilcoxon [45] in PAST4 to examine groupings of dietary consortia by site, date, and crab sex. Principal Components Analysis (PCA) [46, 47] employing the Bray-Curtis dissimilarity index [48, 49] was performed to envision any sample grouping based on diet and to determine which Eukaryotic Families contributed most to differences among samples. Assignment of reads analysis is provided as supplementary material.

Results

Numbers and locations of blue crabs captured in GBE

During 2022 and 2023, the only specimens captured in blue crab pots were those in Oyster River. Across all sites and trap types, 27 blue crabs were captured. In 2022, 18 blue crabs were captured in trapezoidal green crab traps beginning on 2 September and concluding on 19 October, 16 males and 2 females. The first observation of blue crabs was a mated pair captured in a green crab trap deployed on 31 August 2022 at Nannie Island and retrieved 48 h later [11]. The female was immediately post-molt, had shed the gut, and was found to have distended seminal receptacles with sperm plugs [11]. Many crabs (12 of 27) were captured on the degraded oyster grounds at Nannie Island, Moody Point yielded three crabs, two were captured at Fox Point, and one at Cedar Point (Table 1). The largest blue crab captured in a trapezoidal trap was a male from Cedar Point with carapace width of 177 mm. The smallest blue crab was a male with a carapace width of 134 mm at Nannie Island found in a trap with the female (155 mm) with which it had mated. Two additional blue crabs were caught in a crab pot by GBNERR in 2022 in Bunker Creek, both males, one being 188 mm, the largest blue crab captured in this study. In 2023, the first two crabs captured were at Moody Point on 29 August 2023; a mated pair, the female still soft and with sperm plugs [11]. Four additional blue crabs were captured in the trapezoidal green crab traps, three at Moody Point and one at Nannie Island. Of those four crabs, three were male and one was female. Outside the targeted sampling effort, one male blue crab was captured by GBNERR in Oyster River using a crab pot on 7 September, and one male and one female blue crab were captured by NHF&G using a seine in Oyster River on 12 October. Those three were included in the 2023 analyses for a total of 7 blue crabs caught throughout GBE in 2023. The largest 2023 blue crab was a male from Moody Point with a carapace width of 168 mm and the smallest was the female caught at Oyster River with a carapace width of 81 mm. No blue crabs were captured at Fox or Cedar Point in 2023, although farmers reported that blue crabs were seen in these areas.

Metagenomic analysis of diet contents

Metagenomic analysis of shotgun sequencing data from gut contents of blue crabs captured in GBE yielded classified read sets ranging from 207,000 to 2, 900,000 across the 25 samples and identified an average of 590 OTUs per sample. Due to the broad range of reads obtained across gut metagenomes, subsequent statistical analyses used normalized taxon proportions. Eukaryota accounted for an average of 83% of gut assignments (including host), Bacteria for 12% (multiple types), and Virus accounted for roughly 5% (multiple types) of reads (very few Archaea were indicated) (Fig. 4). Excluded from statistical computations were reads assigned to the blue crab family Portunidae (57% of the reads) and the decorator crab family Majidae (16% of reads) as examination of Genus assignment indicated these reads were misclassified Portunidae. Another 36% of reads were removed from the statistical analysis because classification was not provided to Family. Following these exclusions, between 40,000 and 160,000 normalized reads remained available for each blue crab gut sample. Considering diet components that were observed at ≥0.1% of gut reads (≥92% of guts contained the family), most blue crabs had consumed an array of roughly 24 common organisms (Fig. 5). Bonferroni corrected Wilcoxon test p-values showed no difference between the diets of male and female crabs nor among gut contents across sites or between years. Tukey’s pairwise tests indicated that differences among guts were predominantly based on four families that were common to most guts: Enterocytozooa (microsporidian parasite, p = 0), Penaeidae (shrimp, p = 0), Nephropidae (lobster, p = 0.0084), and Pleurotaceae (fungi, p = 0.0085). Principal Component Analysis illustrated that diets did not cluster by site, year, or sex (Fig. 6). Other families identified at 0.1% or higher were parasites and common estuarine organisms such as mussel, sea slug, amphipod, barnacle, marine worm, acorn worm, horseshoe crab, sea spider, fish, coral [66], and gastropod, as well as several terrestrial species (e.g., crayfish, aphid, spider, pillbug, fly, and butterfly) (Table 2). Organisms identified at ~ 0.05–0.1% included, sea anemone, oyster, and additional terrestrial species (e.g., snail, beetle, tick) that commonly are found in diets of nearshore benthic organisms (Table 2). Present but at lower proportions were and several teleost families (Supplemental Table 1 Read count summary for all taxa).

Table 2 The 45 most common eukaryote families other than crab detected in Great Bay Estuary blue crab (Callinectes sapidus) gut metagenomes

Fig. 4

Stacked bar chart showing relative normalized proportions of eukaryote and other domains detected in guts of adult blue crabs captured in GBE. Most specimens had > 50% Eukaryote sequences. This figure includes all reads

Fig. 5

Relative proportions of the 24 most common (≥0.1% of gut reads) food taxa in diets of GBE blue crabs

Fig. 6

Principle Components Analysis of blue crab diets based on the 24 most common organisms

Discussion

Although there have been anecdotal reports of blue crabs in New Hampshire waters prior to 2022 [11], this is the first documentation of frequent blue crab capture in GBE. Blue crab range expansion into the Gulf of Maine system due to warmer waters has been predicted [4] and this study adds evidence to substantiate that prediction, as multiple pairs of mating blue crabs were captured over both years as well as consistent catch over both years throughout the Bay (Table 1). Two mating pairs, one in 2022 [11] and another in 2023 (this report), imply that blue crabs inhabiting GBE are reproducing, and as water warms, spawning can occur earlier [50]. Nevertheless, in this study only adults were captured; no juveniles or gravid females were observed. Juveniles and blue crab larvae have been detected in Southern Maine [67], so similar patterns are possible, albeit not observed, in this southern region. Validation of a self-sustaining breeding population of blue crabs will require detection of planktonic larvae, as well as observation of gravid females and juveniles.

Evaluating the potential effect of blue crabs on oysters is important because GBE oyster populations have declined precipitously over the past decades [51] due to overharvesting, disease, habitat alteration, and predation. When conceiving of this metagenomic study, species of particular interest were green crabs, oysters, tunicates, and oyster drills, as these organisms are associated with oyster restoration in GBE and have been noted as species of concern by the NH Oyster Aquaculture industry [52]. Blue crabs have been known to prey on bivalves [53], and specifically oysters [54, 55] in other studies. Oyster DNA (Crassoatrea) was detected in these gut samples, as was the DNA of gastropods (albeit not oyster drills) and tunicates.

Green crabs (Carcinus) were not detected in any of the blue crab gut samples, even though blue and green crabs were captured in the same traps. Blue crabs have been shown to slow the spread of green crabs and their abundance is less in areas with blue crabs [62]. Thus, although green crabs were not detected in these gut samples, increasing numbers of blue crabs could potentially provide some control of the invasive green crab population, which is thriving in GBE [20,21,22]. Interestingly, Callinectes DNA has appeared in the gut of a green crab captured from the same sampling area as this paper references (data not shown), indicating that some parts of the blue crab, whether they be juvenile, biodeposit, or molt are being consumed by green crabs. The detection of Callinectes DNA in the diet of another species provides additional evidence that blue crabs are expanding into GBE and constitutes another approach for tracking the spread of blue crabs. Other arthropods were detected, specifically the family Varunidae (Hemigrapsus, another invasive crab species), horseshoe crab (Limulus), and lobster (Homarus) (Table 2). Lobster is an important fishery in the Gulf of Maine [57], and horseshoe crabs have an abundance of ecological [58] and biomedical importance [59]. Blue crabs could cause ecosystem shifts and might compete with native crabs, such as the rock crab, Cancer irroratus, that consume barnacles and mussels [60], species shown here to be consumed by blue crabs (Table 2).

Within Eukaryotes, 9% of sequences were for a microsporidian (Enterocytozoonidae), which is a common parasite of shrimp [64], a common organism in these blue crab gut samples. However, another study [65] described a possible new genus of Enterospora within the Enterocytozoonidae family that infected two European crab species. Blue crabs may be displaying this parasite as both a mix from consuming infected shrimp or from infection themselves. The proportion of viral sequences (5% of total) reads is not entirely unusual; viruses are ubiquitous and common in aquatic environments [68, 69] and gut samples [70]. Future studies could focus on viruses specifically with this type of methodology because viruses can affect both farmed and wild crustaceans [71], and with blue crabs being a successful fishery elsewhere, it would be important to investigate how these viruses can affect this economically valuable species. Blue crab diets also included insects of terrestrial origin such as spiders and flies. In most cases, the specimens were captured a few meters from shore, so it is highly likely that the diet of adult blue crabs would include terrestrial components associated with runoff.

Using metagenomics to analyze the diet of blue crabs as they establish viable populations in GBE will be a useful tool for predicting how these range expanding organisms are interacting within this important estuarine ecosystem. It is important to note, the blue crabs captured were in late August to mid-October, so their diet composition may change depending on the time of year, for example if they were captured in the Spring or mid-Summer. Also, although blue crabs are thought to be opportunistic feeders [61] and their diets may be well-known in other areas, this is the first study to investigate what they are consuming in New Hampshire, which is an area that is new to this species. This study provides a framework for investigating how newly introduced species interact in their new environments, in this case Great Bay Estuary. Although results of this gut content study confirm a mixed diet indicative of an opportunistic feeder, there are organisms found in their guts e.g., lobsters and horseshoe crabs, that are of great economic and ecological importance in this area. Investigating if blue crabs are consuming these types of organisms is important. As more blue crabs enter the Bay due to climate change, this diet composition may change so future studies should continue this work and can use this study as guidance. The potential for blue crab predation is critical information for the growing oyster aquaculture industry in New Hampshire and if the risk is high, farmers will need to adjust their cultivation practices to combat the blue crab. This study provides data that are important for predicting how the range expansion of blue crabs may alter how GBE functions as an ecosystem. This type of research promotes the Sustainable Development Goal 14 of Life Below Water by helping end users, such as shellfish industries, to conserve those important and sustainable species for the present and future.