Intact Shrimp Research Articles

Investigating the migration hypothesis: Effects of trypsin-like protease on the quality of muscle proteins of red shrimp (Solenocera crassicornis) during cold storage

This study aimed to investigate the effect of trypsin-like protease (TLP) on the quality of muscle proteins in red shrimp (Solenocera crassicornis) during cold storage. The results indicated that the activity of TLP decreased significantly in the head of shrimp but increased significantly in the muscle tissues during the cold storage. The myofibril fragmentation index (MFI) value of intact shrimp was significantly higher than that of beheaded shrimp, while the Ca2+-ATPase activity of intact shrimp was significantly lower than that of beheaded shrimp. SDS-PAGE analysis showed that the molecular weight of purified TLP from the shrimp head was about 24 kDa, and the TLP showed high activity at 50 °C and pH 8, indicating that the TLP belongs to the trypsin family. Results from in vitro simulation experiments indicated that the process of TLP incubation significantly reduced the particle size and enlarged the distribution of myofibrillar proteins (MPs) in shrimp muscle tissues. The comparisons were made with respect to the control samples. It can be inferred that TLP migrated from the shrimp head to the muscle tissues during storage and thus promoted the degradation of MPs in red shrimp. The beheading treatment could be an effective mean to maintain better quality and extend the commercialization of shrimp products.

Characterization of endogenous enzymes in sword prawn (Parapenaeopsis hardwickii) and their effects on the quality of muscle proteins during frozen storage

Muscle deterioration caused by endogenous enzymes can greatly impact the quality of shrimp products during their processing and storage. In this study, the changes in trypsin, calpain, and cathepsin activity and their effects on muscle proteins in the intact and beheaded shrimp during 120 days of frozen storage were investigated. The results showed that the hardness, stretching force, chewiness, myofibrillar protein (MP) content, and Ca2+-ATPase activity significantly decreased in both the intact and beheaded shrimp samples with the prolonged storage period. Notably, the beheaded shrimp exhibited more stable characteristics than the intact shrimp samples throughout frozen storage. The endogenous enzyme analysis showed that the variations in the trypsin, calpain, and cathepsin activities were significantly pronounced in the intact shrimp compared to those in the beheaded samples. Furthermore, the myofibrillar fragmentation index, SDS-PAGE, and histological structure analysis revealed that the beheading treatment on the shrimp greatly inhibited the dissociation of muscle proteins during frozen storage. Our findings showed that the trypsin in the head of shrimp could be transferred to muscle tissues through the first abdominal segment during long-term storage, thus accelerating the degradation of MPs in the muscle tissues. Therefore, the beheading treatments effectively prolonged the shelf-life of stored shrimp products.

Investigation of the activity of cathepsin B in red shrimp (Solenocera crassicornis) and its relation to the quality of muscle proteins during chilled and frozen storage.

Cathepsin B is a cysteine protease that has important effects on the quality of muscle products. In this study, the changes of cathepsin B activity and its relation to muscle proteins were investigated in intact and beheaded shrimp during chilled and frozen storage. The obtained results indicated that the water holding capacity (WHC), shear force, hardness, and myofibrillar protein (MP) content all significantly decreased in both the intact and beheaded shrimp samples with increasing storage period (p<0.05). Specifically, beheading shrimp exhibited much more stable characteristics than intact shrimp samples during both chilled and frozen storage. The enzyme activity results suggested that cold temperature and storage induced the release of cathepsin B from the lysosomes to the mitochondria, sarcoplasm, and myofibrils in the muscle tissues. Furthermore, SDS-PAGE and transmission electron microscopy (TEM) analysis revealed that beheading the shrimp greatly inhibited the dissociation of shrimp muscle proteins during storage. The current findings suggest that cathepsin B located in the head of shrimp was likely transferred to the muscle through the first abdominal segment during storage, accelerating the dissociation of the muscle proteins. Therefore, beheading the shrimp was conducive to prolonging the shelf-life of stored shrimp products.

Influence of Cold Storage on the Quality of Muscle and the Activity of Cathepsin H in Red Shrimp( Solenocera crassicornis )

To investigate the changes of cathepsin H activity and muscle quality of shrimp (Solenocera crassicornis) under different conditions, the samples were refrigerated at 4 °C for 6 days and frozen at -18 °C for 120 days, respectively. The changes of pH, water retention, hardness, springness, myofibrillar protein content, water content, and cathepsin H activity in intact shrimp and beheaded shrimp were compared and analyzed during chilling storage. Results showed that, with the increasing of storage period, the pH of muscle from the whole shrimps and beheaded shrimps both increased continuously, while the increasing rate of beheaded shrimp was slightly lower than that of whole shrimp samples. The myofibrillar protein (MP) content of both shrimp muscle decreased gradually with the prolonging of storage time. The MP content in intact shrimp and beheaded shrimp group decreased by 38.32% and 30.88% after refrigerated for 6 days, and by 61.67% and 52.09% after frozen for 120 days, respectively. During the whole frozen storage, the hardness and springness of the two groups of shrimps increased first and then decreased, while during the cold storage, the hardness and springness of the two groups of shrimp always decreased. The changes of cathepsin H activity in different subcellular fractions were different. The activity of cathepsin H in frozen condition was lower than that in cold condition, and the activity of cathepsin H in intact shrimp group was higher than that in beheaded shrimp group. The results showed that compared with the whole shrimp, the storage of beheaded shrimp was more conducive to the protection of muscle quality, and the activity of cathepsin H enzyme was greatly affected by the storage temperature. The results would provide a theoretical basis for the changes of physicochemical properties and cathepsin activity of shrimp under different conditions.

Spatial molecular-dynamically ordered NMR spectroscopy of intact bodies and heterogeneous systems

Noninvasive evaluation of the spatial distribution of chemical composition and diffusion behavior of materials is becoming possible by advanced nuclear magnetic resonance (NMR) pulse sequence editing. However, there is room for improvement in the spectral resolution and analytical method for application to heterogeneous samples. Here, we develop applications for comprehensively evaluating compounds and their dynamics in intact bodies and heterogeneous systems from NMR data, including spatial z-position, chemical shift, and diffusion or relaxation. This experiment is collectively named spatial molecular-dynamically ordered spectroscopy (SMOOSY). Pseudo-three-dimensional (3D) SMOOSY spectra of an intact shrimp and two heterogeneous systems are recorded to evaluate this methodology. Information about dynamics is mapped onto two-dimensional (2D) chemical shift imaging spectra using a pseudo-spectral imaging method with a processing tool named SMOOSY processor. Pseudo-2D SMOOSY spectral images can non-invasively assess the different dynamics of the compounds at each spatial z-position of the shrimp’s body and two heterogeneous systems.

Hyperglycemic and osmotic effects of dopamine and recombinant hormone CHH-B1 in the Pacific white shrimp Litopenaeus vannamei

The effects of dopamine (DA) on crustacean hyperglycemic hormone (CHH) release and osmoregulation were investigated in the white shrimp Litopenaeus vannamei. Application of 2 μg of the recombinant CHH-B1His hormone or 2 × 10−6 mol L−1 of DA to intact shrimp caused an increase in the hemolymph glucose levels 1 h post-injection, suggesting that DA might stimulate hyperglycemia through CHH release from the sinus glands. This assumption was supported by similar experiments using bilaterally eyestalk-ablated shrimp. Additionally, rCHH-B1His restored the osmoregulatory capacity (OC) of shrimp under hyperosmotic conditions to basal values 2 h post-injection, whereas DA led to an OC decrease in shrimp at all sampling times. These neuroendocrine factors may be involved in the control of metabolism and osmoregulation in L. vannamei and could be important for its adaptation to different environmental conditions.

Effect of Particle Size on the Kinetic Parameters of the Deproteinization Process of Galactose Supplemented Shrimp Shells by Aspergillus niger

The aim of the research was to evaluate the ability of the fungus Aspergillus niger to carry out the deproteinization of shrimp shells supplemented with galactose as a carbon source and study the effect of the particle size of the shrimp shells on the kinetic parameters of the deproteinization process. Grounding of the shells resulted in a higher specific growth rate of A. niger and enhanced protease production by 2.6 fold. The temperatures of the shrimp shells and exhaust gas declined at the beginning as the heat losses from the bioreactor (due to evaporation) were higher than the heat generated by the metabolic activity. After 24h, the temperature of the shrimp shells and exhaust gas started to rise as a result of increased metabolic activity. Temperature peaks of 38.3 °C and 37.8 °C for the shrimp shells and 29.1 oC and 29.0 oC for the exhaust gas were noticed after 60 and 72 h of deproteinization for the ground and intact shrimp shells, respectively. There were no temperature gradients in the radial or axial direction because of mixing. The pH first decreased with time due to production of acid protease and then increased due to the buffering capacity of the calcium carbonate released from shrimp shells and the production of ammonium nitrogen. A significant reduction in the moisture content was noticed during the deproteinization process. In order to maintain the moisture in the bioreactor at the desired level, the exhaust gas should be passed through a condensation tower and the recovered water be pumped back into the bioreactor through the aeration tube. The galactose concentration decreased with time and the rate of galactose utilization was significantly higher in case of ground shrimp shells. Size reduction results in higher surface area and shorter pathways for nutrients diffusion. The protein removal efficiency (30.45% - 33.23%) did not correspond to the protease production. This could be a result of unsuitable pH and temperature conditions for the hydrolysis of shells proteins. The chitin concentration increased over time from the initial value of 16.59 % to final values of 21.99 %and 22.68% for the intact and ground shells, respectively. The color of the intact and ground shells was pale pink-orange with some tan patches. Some of the ground shrimp shells agglomerated and formed small balls. This problem was not observed with the intact shells. The ground shells had more white precipitants which were believed to be a result of break down of substances from the shell matrix.

Quantitative analysis of hypertrophy and hyperactivity in the androgenic gland of eyestalk-ablated male Pacific white shrimp Litopenaeus vannamei during molt stages

Quantitative analysis of hypertrophy and hyperactivity in tissue of the androgenic gland of eyestalk-ablated male Pacific white shrimp Litopenaeus vannamei was made during the molt cycle by measuring the area of androgenic gland cells located in the subterminal region of the terminal ampoule and measuring the expression of the insulin-like androgenic gland hormone precursor gene (Lv-IAG) in the distal vas deferens and terminal ampoule, respectively. Data were compared to intact shrimp at the same molt stages. In eyestalk-ablated shrimp, the lack of negative control from the X-organ–sinus gland complex produced hypertrophy in androgenic gland cells, because the area of the cells folded approximately 1.5 times up to the intermolt stage, when compared to the intact group. It also produced hyperactivity because the relative expression of Lv-IAG was significantly higher than the intact group at all molt stages. After gathering data from the vas deferens and terminal ampoule, the relative expression of Lv-IAG was maintained at higher levels, without a significant difference during the molt stages. However, after each tissue was analyzed independently, a bell-shape curve pattern occurred, according to the molt cycle, as also occurred in intact shrimp. This suggests that in the absence of eyestalks, the brain and nervous system are operating secondarily to synchronize Lv-IAG expression into the molt cycle. The relative expression of Lv-IAG was significantly greater in the terminal ampoule than in the distal vas deferens. In summary, these findings support a reproductive endocrine axis in L. vannamei, as an eyestalk–androgenic gland linkage concomitant with the molt cycle.

Expression profiles and localization of vitellogenin mRNA and protein during ovarian development of the giant tiger shrimp Penaeus monodon

Ovarian maturation of the giant tiger shrimp (Penaeus monodon) results from rapid synthesis and accumulation of a major yolk protein, vitellin which is derived from its precursor, vitellogenin. The expression profiles and localization of P. monodon vitellogenin 1 (PmVtg1) were examined. PmVtg1 mRNA was expressed only in ovaries and hepatopancreas but not in other tissues of female and testes of male broodstock. In wild intact broodstock, PmVtg1 mRNA was expressed at a low level in stage I ovaries and up-regulated in stages II and III being significantly reduced in stage IV ovaries (P<0.05). In eyestalk-ablated broodstock, the expression level of PmVtg1 mRNA was increased to nearly a peak level in vitellogenic (stage II) ovaries and peaked in cortical rod (stage III) ovaries (P<0.05). The level of PmVtg1 mRNA in hepatopancreas of intact shrimp with stages II and III ovaries was significantly greater than those with stages IV and V (post-spawning) ovaries. Interestingly, PmVtg1 mRNA in hepatopancreas was 25–40 times higher than that in ovaries of intact shrimp with stages I–III of ovarian development. In situ hybridization revealed that PmVtg1 mRNA was clearly localized in the cytoplasm of follicular cells surrounding late previtellogenic, vitellogenic and cortical rod oocytes. Quantitative real-time PCR against different periods of spawned eggs suggested that PmVtg1 mRNA was also synthesized in oocytes. Western blot analysis revealed a similar profile of ovarian P. monodon vitellin and PmVtg1 mRNA during ovarian development and also suggested that the Vtg protein in hepatopancreas and Pm-vitellin in ovaries should have encoded from different Vtg genes. Immunohistochemistry confirmed that Pm-vitellin was localized in developing, vitellogenic and mature oocytes but not in the follicular cells.

Energy balance of spermatophores and sperm viability during the molt cycle in intact and bilaterally eyestalk ablated male Pacific white shrimp Litopenaeus vannamei

The energy levels of spermatophores and sperm viability during the molt cycle were investigated in intact and bilaterally eyestalk ablated male Pacific white shrimp Litopenaeus vannamei. Sexual organs and spermatophores, expressed either as absolute weight or as a somatic index, and sperm counts were significantly higher in the ablated group. A continuous increase in the weight of the vas deferens throughout the molt cycle was observed in the ablated group but not in the control one. This suggests formation of new spermatophores in the vas deferens from the early premolt stage, negatively controlled by hormones from the X organ–sinus gland complex. Spermatophores contained the highest ATP level and adenylate energy charge (AEC), once they were visible in the terminal ampoule after ecdysis at the late postmolt stage of the first molt cycle, where it reached a mean ATP value of 3.3±0.5nmol per spermatophore in the intact group and 5.85±1.4nmol per spermatophore in the ablated group. Both groups had an AEC of ~0.73. ATP and AEC progressively decreased in both groups during the first molt cycle. At the late premolt stage, the intact group had values of ~1.6nmol per spermatophore and ~0.5 respectively, whereas the ablated group had values of ~2.9nmol per spermatophore and ~0.6, respectively. However, after ecdysis at the early postmolt stage of the second molt cycle, ATP and AEC increased in the intact group to ~2.2nmol per spermatophore and ~0.6, respectively, whereas in the ablated group, they decreased to ~1.5nmol per spermatophore and ~0.48, respectively. This differential pattern was also found for arginine phosphate, where it increased in the intact group from 24.8±7.5 to 55.2±5.5nmol per spermatophore, while it decreased in the ablated group from 78.8±10.5 to 59.6±15.5nmol per spermatophore. This suggests a cyclic molt-regulated energy balance, where new spermatophores are energy “recharged” in intact shrimp, while this process is apparently disrupted in ablated shrimp.

Identification of reproduction-related proteins and characterization of the protein disulfide isomerase A6 cDNA in ovaries of the giant tiger shrimp Penaeus monodon

Proteomic analysis was carried out for identification of proteins functionally involved in ovarian development of the giant tiger shrimp (Penaeus monodon). A total of 335 protein spots including 183 spots from vitellogenic (stage II) and 152 spots from mature (stage IV) ovaries of intact P. monodon broodstock were examined. Of these, 75 (40.98%) and 59 (38.82%) spots significantly matched known proteins in the databases, respectively. In addition, 270 protein spots including 167 and 103 spots from respective ovarian stages of eyestalk-ablated broodstock were also characterized. A total of 95 (56.89%) and 62 (60.19%) spots matched known proteins, respectively. Among differentially expressed reproduction-related proteins, the full-length cDNA of protein disulfide isomerase A6 (PmPDIA6) was further characterized by RACE-PCR. PmPDIA6 was 1946bp in length containing an open reading frame (ORF) of 1293bp corresponding to a polypeptide of 430 amino acids. PmPDIA6 was up-regulated at stage III ovaries in intact shrimp (P<0.05). Interestingly, eyestalk ablation resulted in a lower expression level of PmPDIA6 in each stage of ovarian development compared to that of intact broodstock (P<0.05). Results in this study clearly indicated the potential of cellular proteomic studies and gene expression analysis for identification of proteins/genes differentially expressed during ovarian development of P. monodon.

PAH effects on grass shrimp, Palaeomonetes pugio: interactions with the ecdysteroid system

The steroid hormone ecdysone controls molting and differentiation in Arthropods. We used a multi-tiered approach to investigate whether polycyclic aromatic hydrocarbons (PAHs) interact with the functional Ecdysone Receptor (EcR) in vitro, and whether PAHs have molting or reproductive effects in vivo. We focused on four PAHs: pyrene, benzo[a]pyrene, benzo[b]fluoranthene, and chrysene. A reporter gene assay using a Chinese hamster ovary cell line which stably expresses the EcR/RXR complex, showed that the four PAHs had no agonistic activity, but were able to enhance reporter gene expression when given in conjuction with an ecdysteroid agonist, muristerone A. An ecdysone-responsive Drosophila cell line (Cl 8+) showed that PAHs alone had no effect, but were able to enhance the differentiation response in the presence of muristerone A. Finally, ablated grass shrimp ( Palaeomonetes spp.) were exposed to 100 nM chrysene, and molted approximately 12 h earlier than the vehicle controls. Since chrysene was able to enhance reporter gene activation and the Cl 8+ cellular response in the presence of an ecdysteroid, the accelerated molting correlates well with the in vitro assays. A chronic 8 week exposure study on intact shrimp is currently underway to determine molting and reproductive endpoints, as well as P450 and lipovitellin levels.

Role of Mechanosensory Stimuli in Intraspecific Agonistic Encounters of the Snapping Shrimp (Alpheus heterochaelis).

Intraspecific agonistic encounters in snapping shrimp (Alpheus heterochaelis) were analyzed using single-frame video analysis. The pair of conspecifics in such an encounter are designated as the snapper, which emits a fast water jet by very rapid closure, or snap, of the large modified snapper claw, and the receiver, which is the target of the water jet. The behavior of both snapper and receiver was evaluated before, during, and after the snap. Interactions between two intact shrimp (experimental series I) were compared with those between an intact shrimp and a "deprived" opponent (one with mechanosensory occlusion produced by coating the setae on the snapper claw with clear lacquer) (experimental series II). The behavior of the receiver is significantly changed by the occlusion, but that of the snapper is not. Intact and deprived opponents usually face each other during snapping, which is often preceded by touching of frontal appendages. The mean duration of claw cocking before snapping is about 500 ms. More than 50% of all snaps (and especially initial snaps) are directed towards the opponent, the water jet usually hitting the snapper claw of the receiver from a mean distance of 0.9 cm. Male shrimp show longer cocking durations, keep a shorter distance, and hit their opponents more often than do females. Intact and deprived snappers usually retreat immediately after snapping, but intact receivers usually approach. In contrast, deprived receivers retreat in most cases and show significantly prolonged latencies compared to intact receivers. Thus, mechanoreceptors on the snapper claw of the receiver play a significant role in intraspecific agonistic encounters.

Automated Headspace Ammonia Analysis of Shrimp

Determination of shrimp odor by sensory evaluation is subjective, difficult to quantify, or depends on complex and time consuming chemical analyses. Ammonia (NH3) is the main off-odor of shrimp. This study developed a methodology for repeatable, objective and rapid headspace NH3 analysis of shrimp. An electrode was connected to a computer to quantify NH3 in the headspace of intact shrimp kept in an air-tight box. The only sample preparation was to spray NaOH on shrimp. Less than two minutes were needed to obtain results. Ammonia levels measured by this method were comparable to that of wet chemical methods. A sensory panel determined that the rejection level of NH3 in shrimp was 230 ppm. This procedure could be used as a fast, simple and objective method to evaluate shrimp odor quality.

YOLK PROTEIN PRODUCTION IN THE MARINE SHRIMP PENAEUS VANNAMEI

ABSTRACT Yolk production of the marine shrimp Penaeus vannamei was measured using an antibody to a subunit of the yolk protein complex. The effects of unilateral eyestalk ablation on protein synthesis of the hepatopancreas and ovary were measured in vitro. Initially, activity in both tissues increased; 21 days after eyestalk ablatior activity levels in both tissues returned to levels equal to or lower than preablation levels. The yolk protein content of the tissues increased shortly after eyestalk ablation and reached a maximum of 8% in ovarian tissue, but less than 1% in the hepatopancreas. Yolk proteins in the hemolymph were detected using an enzymelinked immunoassay. Both intact controls and eyestalk-ablated shrimp had as much as 1 mg/ ml yolk protein in the hemolymph during ovarian maturation. Newly released shrimp eggs had an average of 53 ng yolk protein per egg. Eyestalk ablation induced a rapid increase in protein synthesis in both ovary and hepatopancreas. During ovarian maturation, yolk proteins were transported through the hemolymph in this marine shrimp.

Effects of monoamines and monoaminergic agents on migration of the red pigment in chromatophores of the shrimp, Macrobrachium ohione

1. The effects of 5-hydroxytryptamine (5-HT) and the 5-hydroxytryptaminergic agents fenfluramine, fluoxetine and 5-MeODMT, and of dopamine (DA) and the dopaminergic agents ADTN, apor-morphine and piribedil on the disposition and translocation of the red pigment in chromatophores of the shrimp, Macrobrachium ohione, were determined. 2. 5-HT evoked dose-dependent red pigment dispersion whereas DA produced dose-related concentration of the red pigment, but only in intact shrimp. Neither of these monoamines affected red pigment migration in vitro. 3. The data obtained using drugs known to act selectively in mammals via 5-hydroxytryptaminergic and dopaminergic mechanisms on the translocation of the red pigment from the chromatophores both in vitro and in vitro lead to the hypothesis that 5-HT and DA serve as neurotransmitters triggering release of red pigment-dispersing hormone and red pigment-concentrating hormone, respectively, in the shrimp, Macrobrachium ohione.

POSTMORTEM QUALITY CHANGES IN ICED PACIFIC SHRIMP (Pandalus jordani)

The postmortem biochemical changes in intact raw shrimp during iced storage were investigated and the relationship between these changes and the quality of the cooked product was evaluated. Changes in pH, microbial numbers, total and nonprotein nitrogen, tyro‐sine and carotenoid pigment content, proteolytic and polyphenolase enzyme activities and levels of trimethylamine‐oxide, trimethylamine, dimethylamine and formaldehyde were determined. Chemical changes were shown to be mediated by a combination of bacterial action and endogenous enzymatic activity. The washing action of melting ice could have played a major role. Organoleptic quality as shown by flavor panel evaluations progressively declined during an 8‐day storage period. Dimethylamine and formaldehyde levels in intact shrimp progressively increased as flavor panel scores decreased and may offer additional sensitive indices of cooked meat quality