Identifying the genes involved in the egg-carrying ovigerous hair development of the female blue crab Callinectes sapidus: transcriptomic and genomic expression analyses.

BACKGROUND: Crustacean female sex hormone (CFSH) controls gradually developing adult female-specific morphological features essential for mating and brood care. Specifically, ovigerous hairs are developed during the prepuberty molt cycle of the blue crab Callinectes sapidus that are essential for carrying the eggs until they finish development. Reduced CFSH transcripts by CFSH-dsRNA injections result in fewer and shorter ovigerous hairs than the control. This study aimed to identify the specific genes responsible for ovigerous hair formation using transcriptomic, genomic and expression analyses of the ovigerous setae at three stages: prepuberty at early (OE) and late premolt (OL), and adult (AO) stages. RESULTS: The de novo Trinity assembly on filtered sequence reads produced 96,684 Trinity genes and 124,128 transcripts with an N50 of 1,615 bp. About 27.3% of the assembled Trinity genes are annotated to the public protein sequence databases (i.e., NR, Swiss-Prot, COG, KEGG, and GO databases). The OE vs. OL, OL vs. AO, and OE vs. AO comparisons resulted in 6,547, 7,793, and 7,481 differentially expressed genes, respectively, at a log2-fold difference. Specifically, the genes involved in the Wnt signaling and cell cycle pathways are positively associated with ovigerous hair development. Moreover, the transcripts of ten cuticle protein genes containing chitin-binding domains are most significantly changed by transcriptomic analysis and RT-qPCR assays, which shows a molt-stage specific, down-up-down mode across the OE-OL-AO stages. Furthermore, the expression of the cuticle genes with the chitin-binding domain, Rebers and Riddiford domain (RR)-1 appears at early premolt, followed by RR-2 at late premolt stage. Mapping these 10 cuticle protein sequences to the C. sapidus genome reveals that two scaffolds with a 549.5Kb region and 35 with a 1.19 Mb region harbor 21 RR1 and 20 RR2 cuticle protein genes, respectively. With these findings, a putative mode of CFSH action in decapod crustaceans is proposed. CONCLUSIONS: The present study describes a first step in understanding the mechanism underlying ovigerous hair formation in C. sapidus at the molecular level. Overall, demonstrating the first transcriptome analysis of crustacean ovigerous setae, our results may facilitate future studies into the decapod female reproduction belonging to the suborder Pleocyemata.

Common fear molecules induce defensive responses in marine prey across trophic levels.

Predator-prey interactions are a key feature of ecosystems and often chemically mediated, whereby individuals detect molecules in their environment that inform whether they should attack or defend. These molecules are largely unidentified, and their discovery is important for determining their ecological role in complex trophic systems. Homarine and trigonelline are two previously identified blue crab (Callinectes sapidus) urinary metabolites that cause mud crabs (Panopeus herbstii) to seek refuge, but it was unknown whether these molecules influence other species within this oyster reef system. In the current study, homarine, trigonelline, and blue crab urine were tested on juvenile oysters (Crassostrea virginica) to ascertain if the same molecules known to alter mud crab behavior also affect juvenile oyster morphology, thus mediating interactions between a generalist predator, a mesopredator, and a basal prey species. Oyster juveniles strengthened their shells in response to blue crab urine and when exposed to homarine and trigonelline in combination, especially at higher concentrations. This study builds upon previous work to pinpoint specific molecules from a generalist predator's urine that induce defensive responses in two marine prey from different taxa and trophic levels, supporting the hypothesis that common fear molecules exist in ecological systems.

Investigating conspecific CsRV1 transmission in Callinectes sapidus.

A reo-like virus, CsRV1, is found in blue crabs, Callinectes sapidus, from every North American location assessed, including Chesapeake Bay and the Atlantic and Gulf coasts, USA and associated with blue crabs in softshell production. CsRV1-associated crab mortality is prevalent in captive crabs, but it is still unknown how CsRV1 is transmitted. The purpose of this study was to examine the role that conspecific predation or scavenging may play in per os transmission in single exposure and repeated exposure experiments. For viruses without cell culture propagation, repeated exposure experiments have the challenge of presenting the virus consistently during the experiment and across time replicates. In a single-exposure experiment, none of the crabs fed muscle tissue of crabs carrying intense infections of CsRV1 developed CsRV1 infections. In a repeated-exposure trial, using infected muscle tissue prepared in alginate blocks, CsRV1 was detected in 11% of the crabs fed infected tissue but was not significantly different from the control group fed alginate lacking CsRV1. For repeated per os exposure experiments, the study demonstrated the utility of using alginate to present the same homogenous sample of virus, both injected and per os, over time for oral challenge experiments. Conspecific predation and scavenging could be a transmission route, but future work into this and other possible routes of transmission for CsRV1 is important to better understand the role this virus plays in wild crab populations and the soft-shell crab industry.

White spot syndrome virus (WSSV) in cultured juvenile blue crabs Callinectes sapidus: oral versus injection exposure, and feeding frequency effects.

The efficacy of oral versus injection exposure and the effect of feeding frequency on the transmission of white spot syndrome virus (WSSV) in cultured juvenile blue crabs Callinectes sapidus were investigated. Crabs in Group 1 (G-1, n = 48) were exposed once orally to 100 mg of WSSV-infected shrimp tissue mg-1 of body weight (BW). The oral inoculum contained 2.6 × 109 WSSV genome copies mg-1 tissue. Group 2 (G-2, n = 46) received the same dosage once weekly for 5 wk. Group 3 (G-3, n = 12) was injected with 0.01 ml (2.6 × 107 genome copies 0.01 ml-1) WSSV inoculum g-1 BW. Group 4 (G-4, n = 12) was injected with 0.01 ml WSSV-negative shrimp serum and saline mixture g-1 BW. Dead and moribund animals were frozen at -80°C. After 37 d, all remaining crabs were frozen. Genomic DNA from gill tissue was evaluated for the presence and quantity of WSSV using TaqMan real-time PCR. All G-3 animals died and tested positive. No G-4 animals died or tested positive. In the fed groups, WSSV prevalence was approximately 16%, but viral load was higher and survival was lower in G-2 compared to G-1. Injected animals carried a higher viral load than fed animals, and dead animals had higher viral loads than live animals. Blue crab juveniles are susceptible to WSSV, but oral exposure does not efficiently transmit WSSV in juvenile blue crabs. Some animals can die from WSSV if repeatedly exposed.

Implications on the Pb bioaccumulation and metallothionein levels due to dietary and waterborne exposures: The Callinectes danae case.

This study aimed to assess the bioaccumulation of Pb and induction of metallothionein-like proteins (MT) in Callinectes danae through single and combined dietary and waterborne exposures. Male C. danae individuals were collected in the south area of the Cananéia-Iguape-Peruíbe Protected Area (APA-CIP), in São Paulo State, Brazil. After an acclimatization period, exposure assays were performed during 7 and 14 days, at two Pb concentrations (0.5 e 2.0 µg/g) in 4 treatments: 1) control; 2) contaminated water only; 3) contaminated food only; 4) contaminated water and food. The results indicate that C. danae is highly tolerant to Pb exposure at the evaluated concentrations. In gills, Pb bioaccumulation is more dependent of water efflux and time of exposure (higher Pb values). However, pathways act simultaneously in the induction of MT expression in this tissue. The decreases in Pb accumulation in the combined treatments and MT increases after 14 days in gills suggests that these proteins play a detoxification function in the presence of Pb. In hepatopancreas, depending on the predominance of a certain pathway or combined pathways, accumulation occured at different times. For muscle tissue, bioaccumulation was observed due to contaminated water exposure, but not dietary exposure, probably because Pb concentrations were low.

Habitat fragmentation differentially affects trophic levels and alters behavior in a multi-trophic marine system.

Seagrass, an important subtidal marine ecosystem, is being lost at a rate of 110 km2 year-1, leading to fragmented seagrass seascapes. Habitat fragmentation is predicted to affect trophic levels differently, with higher trophic levels being more sensitive, stressing the importance of a multi-trophic perspective. Utilizing the trophic relationship between the blue crab (Callinectes sapidus) and hard clam (Mercenaria mercenaria), where adult blue crabs prey on juvenile blue crabs, and juvenile blue crabs prey on small hard clams, we examined whether predation rates, abundance, and behavior of predators and prey differed between continuous and fragmented seagrass in a multi-trophic context at two sites in Barnegat Bay, NJ. We tested the hypothesis that fragmented habitats would differentially affect trophic levels within a tri-trophic system, and our results supported this hypothesis. Densities of adult blue crabs were higher in fragmented than continuous habitats. Densities of juvenile blue crabs, the primary predator of hard clams, were lower in fragmented habitats than continuous, potentially due to increased predation by adult blue crabs. Clams experienced lower predation and burrowed to a shallower depth in fragmented habitats than in continuous habitat, likely due in part to the low densities of juvenile blue crabs, their primary predator. Our results suggest that while trophic levels are differentially affected, the impact of habitat fragmentation may be stronger on intermediate rather than top trophic levels in some marine systems.

Sublethal Toxicity of Crude Oil Exposure in The Blue Crab, Callinectes sapidus, at Two Life History Stages.

The 2010 Deepwater Horizon oil spill occurred during peak spawning season for many Gulf of Mexico fish and invertebrates. Early life stages of important fishery species were at risk to encounter crude oil. In this study, we investigated the effect of crude oil exposure on two life stages of the blue crab (Callinectes sapidus). We tested the effects of oil exposure on the survival and growth of larval (pelagic) and juvenile (estuarine) blue crabs as well as the effects of oil on the molt frequency of juveniles. Larval crabs exposed to crude oil showed no discernable growth or mortality differences when compared to non-exposed controls. Juvenile crabs exposed to oil also showed no differences in size but exhibited increased intermolt duration (time between molts). Our study suggests that different life-stages may respond differently when exposed to crude oil and that oil exposure negatively affects growth rate of juvenile blue crabs.

Elevated water temperature increases the levels of reo-like virus and selected innate immunity genes in hemocytes and hepatopancreas of adult female blue crab, Callinectes sapidus.

Seasonal changes in water temperature directly affect the aquatic ecosystem. The blue crab, Callinectes sapidus, inhabiting the Chesapeake Bay has been adapted to seasonal changes of the environmental conditions. In this, the animals halt their physiological process of the growth and reproduction during colder months while they resume these processes as water temperatures increase. We aimed to understand the effect of the elevated temperatures on a disease progression of reo-like virus (CsRLV) and innate immunity of adult female C. sapidus. Following a rise in water temperature from 10 to 23 °C, CsRLV levels in infected crabs rose significantly in hemocytes and multiple organs. However, in hemocytes, the elevated temperature had no effect on the levels of three innate immune genes: Cas-ecCuZnSOD-2, CasPPO and CasLpR three carbohydrate metabolic genes: CasTPS, CasGlyP; and CasTreh and the total hemocyte counts (THC). Interestingly, the hemocytes of CsRLV infected animals exposed to 23 °C for 10 days had significantly elevated levels of Cas-ecCuZnSOD-2 and CasTPS, compared to those of the uninfected ones also exposed to the same condition and compared to hatchery-raised females kept at 23 °C. Despite the lack of changes in THC, the types of hemocytes from the animals with high CsRLV levels differed from those of uninfected ones and from hatchery animals kept at 23 °C: CsRLV-infected crabs had hemocytes of smaller size with less cytosolic complexity than uninfected crabs. It therefore appears that the change in temperature influences rapid replication of CsRLV in all internal tissues examined. This implies that CsRLV may have broad tissue tropism. Interestingly, the digestive tract (mid- and hindgut) contains significantly higher levels of CsRLV than hemocytes while hepatopancreas and ovary have lower levels than hemocytes. Innate immune responses differ by tissue: midgut and hepatopancreas with upregulated Cas-ecCuZnSOD-2 similar to that found in hemocytes. By contrast, hepatopancreas showed a down-regulated CasTPS, suggesting carbohydrate stress during infection.

Larval connectivity of the invasive blue crabs Callinectes sapidus and Portunus segnis in the Mediterranean Sea: A step toward improved cross border management.

The two invasive blue crabs, Callinectes sapidus and Portunus segnis have spread rapidly in the Mediterranean and no data exists on the connectivity of populations. Determining the source and recruitment areas is crucial to prioritize where population control measures should be put into immediate action. We simulated the dispersal of blue crab larvae using a Lagrangian model coupled at high resolution to estimate the potential connectivity of blue crab populations over a 3-year period. Our results reveal that the main areas at risk are the Spanish, French, Italian Tyrrhenian and Sardinian coasts for Callinectes sapidus with high populations connectivity. Tunisia and Egypt represent high auto recruitment zones for Portunus segnis restricted to the central and western basins. This study provides an overview of the connectivity between populations and will help define priority areas that require the urgent implementation of management measures.

Predator signaling of multiple prey on different trophic levels structures trophic cascades.

The capacity of an apex predator to produce nonconsumptive effects (NCEs) in multiple prey trophic levels can create considerable complexity in nonconsumptive cascading interactions, but these effects are poorly studied. We examined such effects in a model food web where the apex predator (blue crabs) releases chemical cues in urine that affect both the intermediate consumer (mud crabs seek shelter) and the basal prey (oysters are induced to grow stronger shells). Shelter availability and predator presence were manipulated in a laboratory experiment to identify patterns in species interactions. Then, experimentally induced and uninduced oysters were planted across high-quality and low-quality habitats with varying levels of shelter availability and habitat heterogeneity to determine the consistency of these patterns in the field. Oyster shell thickening in response to blue crab chemical cues generally protected oysters from mud crab predation in both the laboratory and in field environments that differed in predation intensity, structural complexity, habitat heterogeneity, and predator composition. However, NCEs on the intermediate predator (greater use of refugia) opposed the NCEs on oyster prey in the interior of oyster reefs while still providing survival advantages to basal prey on reef edges and bare substrates. Thus, the combined effects of changing movement patterns of intermediate predators and morphological defenses of basal prey create complex, but predictable, patterns of NCEs across landscapes and ecotones that vary in structural complexity. Generalist predators that feed on multiple trophic levels are ubiquitous, and their potential effects on NCEs propagating simultaneously to different trophic levels must be quantified to understand the role of NCEs in food webs.

Ultrastructure and histochemistry of the male reproductive system of the genus Callinectes Stimpson, 1860 (Brachyura: Portunidae).

We described the ultrastructure and histochemistry of the reproductive system of five Callinectes species, and evaluate the seasonal variation in weight of the reproductive system and hepatopancreas by comparing annual changes of somatic indices. The somatic indices changed little throughout the year. In Callinectes, spermatogenesis occurs inside the lobular testes and, within each lobule, the cells are at the same developmental stage. Spermatogenesis and spermiogenesis follow the same development pattern in all Callinectes studied. Mature spermatozoa are released into the seminiferous ducts through the collecting ducts. Cells of the vas deferens are secretory as evidenced by rough endoplasmic reticulum, Golgi complex, and secretory vesicles that produce the seminal fluid. The anterior vas deferens shows two portions: proximal and distal. In proximal portion (AVDp), spermatozoa are clustered and embedded in an electron-dense, basophilic glycoproteinaceous secretion Type I. In the distal portion (AVDd), the spermatophore wall is formed by incorporation of a less electron-dense glycoproteinaceous secretion Type II. The secretion Type I change to an acid polysaccharide-rich matrix that separates the spermatophores from each other. The median vas deferens (MVD) stores the spermatophores and produces the granular glycoproteinaceous seminal fluid. The posterior vas deferens (PVD) has few spermatophores. Its epithelium has many mitochondria and the PVD seminal fluid changes into a liquid and homogeneous glycoprotein. Many outpocketings in the PVD and MVD help to increase the fluid production. Overall, the reproductive pattern of Callinectes is similar to other species that produce sperm plugs. The secretions of AVD, MVD, and PVD are responsible for the polymerization that forms the solid, waxy plug in the seminal receptacle. The traits identified here are common to all Portunidae species studied so far.

New route of chitosan extraction from blue crabs and shrimp shells as flocculants on soybean solutes.

The chitosan extracted from blue crabs and shrimp shells using calcium oxide (deproteinization) followed by deacetylation which eliminated the demineralization step to reduce the chemical usage and environmental protection. The extracted chitosan examined the flocculation to soybean solutes. The optical density (OD), solid%, and purity% (carbohydrates/soluble solids) after flocculation were measured. The OD was significantly decreased from 0.76 to 0.16 with blue crabs and 0.06 with shrimp shells chitosan-acetate dosing (0.5 g/L). The removal of about 68 and 66% solids was achieved by the addition of 0.5 g/L chitosan-acetate. The purity% was reached about 80% with blue crabs, and 78% with shrimp shells chitosan-acetate. The results of this study verified that the calcium oxide treatment should remove protein and increase the chitin extraction yield on blue crab and shrimp shells. This new route of chitosan extraction should be a useful method for making flocculants in the soybean solutes.

Turbidity interferes with foraging success of visual but not chemosensory predators.

Predation can significantly affect prey populations and communities, but predator effects can be attenuated when abiotic conditions interfere with foraging activities. In estuarine communities, turbidity can affect species richness and abundance and is changing in many areas because of coastal development. Many fish species are less efficient foragers in turbid waters, and previous research revealed that in elevated turbidity, fish are less abundant whereas crabs and shrimp are more abundant. We hypothesized that turbidity altered predatory interactions in estuaries by interfering with visually-foraging predators and prey but not with organisms relying on chemoreception. We measured the effects of turbidity on the predation rates of two model predators: a visual predator (pinfish, Lagodon rhomboides) and a chemosensory predator (blue crabs, Callinectes sapidus) in clear and turbid water (0 and ∼100 nephelometric turbidity units). Feeding assays were conducted with two prey items, mud crabs (Panopeus spp.) that rely heavily on chemoreception to detect predators, and brown shrimp (Farfantepenaus aztecus) that use both chemical and visual cues for predator detection. Because turbidity reduced pinfish foraging on both mud crabs and shrimp, the changes in predation rates are likely driven by turbidity attenuating fish foraging ability and not by affecting prey vulnerability to fish consumers. Blue crab foraging was unaffected by turbidity, and blue crabs were able to successfully consume nearly all mud crab and shrimp prey. Turbidity can influence predator-prey interactions by reducing the feeding efficiency of visual predators, providing a competitive advantage to chemosensory predators, and altering top-down control in food webs.

The smell of success: the amount of prey consumed by predators determines the strength and range of cascading non-consumptive effects.

We examined whether chemically mediated risk perception by prey and the effects of changes in prey behavior on basal resources vary as a function of the amount of prey biomass consumed by the predator. We studied these issues using a tritrophic system composed of blue crabs, Callinectes sapidus (top predator), mud crabs Panopeus herbstii (intermediate prey), and oysters Crassostrea virginica (basal resource). Working in a well characterized field environment where experiments preserve natural patterns of water flow, we found that biomass consumed by a predator determines the range, intensity and nature of prey aversive responses. Predators that consume large amounts of prey flesh more strongly diminish consumption of basal resources by prey and exert effects over a larger range (in space and time) compared to predators that have eaten less. Less well-fed predators produce weaker effects, with the consequence that behaviorally mediated cascades preferentially occur in refuge habitats. Well-fed predators affected prey behavior and increased basal resources up to distances of 1-1.5 m, whereas predators fed restricted diet evoked changes in prey only when they were extremely close, typically 50 cm or less. Thus, consumptive and non-consumptive effects may be coupled; predators that have a greater degree of predatory success will affect prey traits more strongly and non-consumptive and consumptive effects may fluctuate in tandem, with some lag. Moreover, differences among predators in their degree of prey capture will create spatial and temporal variance in risk cue availability in the absence of underlying environmental effects.

The impact of limb autotomy on mate competition in blue crabs Callinectes sapidus Rathbun.

This study is the first to demonstrate experimentally that autotomy (self-amputation of a body part) adversely affects competition for mates. Experiments were conducted using blue crabs Callinectes sapidus Rathbun to examine the consequences of limb loss and pairing precedence on mate acquisition by males. Two adult males of equivalent size were introduced sequentially into pools containing a sexually-receptive female and observed after 24 h and 48 h. One male in each pair was left intact, while the other experienced: (1) no autotomy, (2) autotomy of one cheliped, or (3) autotomy of both chelipeds, one walking leg, and one swimming leg. In the absence of a competitor (first 24 h), both intact and injured males established precopulatory embraces with females. Intact males were highly successful (84-95%) in defending females from intact or injured intruders in the second 24 h period. Both autotomy treatments, however, significantly reduced the ability of males to defend females from intact intruders. Females in experiments suffered greater frequency of limb loss than did males. In the field, paired blue crabs showed significantly higher incidence of limb loss than unpaired crabs. Limb loss frequency increases with body size, and field observations indicated that larger males may be more successful than smaller males in obtaining females. Both experimental manipulations and field studies provide strong evidence for mate competition in this ecologically and commercially important portunid species.