Article

Effect of starvation and refeeding on the hepatopancreas of whiteleg shrimp Penaeus vannamei (Boone) using computer-assisted image analysis

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Abstract

Under normal farming conditions, shrimp can experience starvation periods attributable to disease outbreaks or adverse environmental conditions. Starvation leads to significant morphological changes in the hepatopancreas (HP), being the main organ for absorption and storage of nutrients. In the literature, limited research has described the effect on the HP of periods of starvation followed by refeeding and none in whiteleg shrimp (Penaeus vannamei) using computer-assisted image analysis (CAIA). This study describes the effect of starvation and starvation followed by refeeding on the HP of whiteleg shrimp using CAIA. Visiopharm� software was used to quantify the following morphological parameters, measured as ratio to the total tissue area (TLA): total lumen area (TLA:TTA), haemocytic infiltration area in the intertubular spaces (HIA:TTA), B-cell vacuole area (VBA:TTA), lipid droplet area within R cells (LDA:TTA) and F-cell area (FCA:TTA). Significant changes were measured for HIA:TTA and LDA:TTA during starvation (increase in HIA:TTA associated with decrease in LDA:TTA) and starvation followed by refeeding (decrease in HIA: TTA associated with increase in LDA:TTA). In the future, HIA:TTA and LDA:TTA have the potential to be used in a pre-emptive manner to monitor the health of the HP, facilitate early diagnosis of diseases and study the pathophysiology of the organ.

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... Moreover, with the gradual increase in environmental temperature during this period, the body's metabolic rate and energy demands increase. Hepatopancreas is the metabolic organ that possesses significant functions, such as absorption, storage, metabolism of nutrients such as fat and glucose, and vitellogenin synthesis in crustaceans (Cervellione et al., 2017). The metabolism of nutrients in the hepatopancreas eventually has been converted into energy release; therefore, the hepatopancreas is also regarded as the centre of energy metabolism and the carrier of energy reserve. ...
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The digestive gland of crustacean is involved in various metabolic activities, including the synthesis and secretion of digestive enzymes that begin the process of food digestion, intracellular digestion and absorption of nutrients, storage of reserves, and disposal of waste products. It consists of two glandular lobes which extensively subdivide to form a complex of blind-ending tubules, whose size, surface area, and digestive cells are associated with intracellular digestion and the nutritional status of the organism. The aim of this paper was to study the morphology of the digestive gland in various lobster species and calculate the surface area of tubules, lumen and digestive cells (R-, F-, and B-cells) and their ratios to total tubule surface area. The similarity in ratios obtained in this study between individual lobsters suggests that the method developed in this study can be successfully applied to a range of species. This study describes a novel image processing algorithm for the automatic measurement of the hepatopancreas structure using stained cross sections of digestive gland tubules. The proposed new methodology could be used for studying the physiology and nutrient metabolism of lobsters and other crustaceans. The computer-aided analysis described in this paper is accurate for the quantitative assessment of the lobster's digestive gland structure.
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Physiological cell turnover plays an important role in maintaining normal tissue function and architecture. This is achieved by the dynamic balance of cellular regeneration and elimination, occurring periodically in tissues such as the uterus and mammary gland, or at constant rates in tissues such as the gastrointestinal tract and adipose tissue. Apoptosis has been identified as the prevalent mode of physiological cell loss in most tissues. Cell turnover is precisely regulated by the interplay of various endocrine and paracrine factors, which modulate tissue and cell-specific responses on proliferation and apoptosis, either directly, or by altering expression and function of key cell proliferative and/or death genes. Although recent studies have provided significant information on specific tissue systems, a clearly defined pathway that mediates cell turnover has not yet emerged for any tissue. Several similarities exist among the various tissues with regard to the intermediates that regulate tissue homeostatis, enabling a better understanding of the general mechanisms involved in the process. Here we review the mechanisms by which hormonal and cytokine factors mediate cell turnover in various tissues, emphasizing common themes and tissue-specific differences.
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In this paper, we review the current knowledge about the usage of carbohydrates, lipids and proteins as energy source by marine crustaceans during starvation. Crustaceans are a large and diverse group including some economically important species. The efforts to culture them for human consumption has prompted the interest to understand the preferences of energy sources to be applied for feed formulation and cost reduction. Important differences have been found among species and appear to be related not only to the biochemistry and physiology of nutrition, but also to the living environment of the crustaceans. Furthermore, crustaceans undergo morphological, physiological and behavioral changes due to their natural growing process that affect their feeding habits, an aspect that should be carefully considered. We discuss the current information on marine crustaceans about energy usage and describe areas of future research, where starvation studies render important insights.
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Despite the increasing use of novel molecular techniques in pathology, histology remains the standard method for monitoring tissue alterations and for assessing pathology. Histopathological evaluation is generally laborious and subjective with risk of discrepancies in semi-quantitative scoring between pathologists. In contrast, computer-assisted image analysis (CAIA) is potentially faster, more objective and thus suitable for routine screening. Limited research has been carried out on CAIA in crustacean histopathology, and the methods described were not fully automated. Therefore, the aim of this study was to develop CAIA in whiteleg shrimp (Penaeus vannamei) for the study of the hepatopancreas. Paraffin sections were immunohistochemically stained with monoclonal antibodies WSH8 against haemocytes and counterstained with Mayer′s haematoxylin for detection of haemocytes and B-cell vacuoles, and modified toluidine blue protocol was used for detection of F-cells; frozen sections were stained with Oil Red O for detection of lipid droplets within R-cells. Visiopharm® software was used to develop and validated protocols for the quantification of morphological parameters (areas of haemocyte infiltration, F-cells, B-cell vacuoles, lipid droplets and their ratios to total tissue area and total lumen area). These protocols enable the future use of CAIA for determination of the nutritional and pathological condition of this organ.
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Pathology in penaeid shrimps relies on histology, which is subjective, time-consuming and difficult to grade in a reproducible manner. Automated image analysis is faster, objective and suitable for routine screening; however, it requires standardized protocols. The first critical step is proper fixation of the target tissue. Bell & Lightner's (A Handbook of Normal Penaeid Shrimp Histology, 1988, The World Aquaculture Society, Baton Rouge) fixation protocol, widely used for routine histology of paraffin sections, is not optimized for image analysis, and no protocol for frozen sections is described in the available literature. Therefore, the aim of this study was to optimize fixation of the hepatopancreas (HP) from whiteleg shrimp (Penaeus vannamei) for both paraffin and frozen sections using a semiquantitative scoring system. For paraffin sections, four injection volumes and three injection methods were compared, for frozen sections, four freezing methods and four fixation methods. For paraffin sections, optimal fixation was achieved by increasing threefold the fixative volume recommended by Bell and Lightner, from 10% to 30% of the shrimp body weight, combined with single injection into the HP. Optimal fixation for frozen sections was achieved by freezing the cephalothorax with liquid nitrogen, followed by fixation of the section with 60% isopropanol. These optimized methods enable the future use of image analysis and improve classical histology.
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The aim of this study was to analyze the histology and the histochemical distribution of glycoproteins (GPs) and lipids of the hepatopancreas of Cyrtograpsus angulatus and Neohelice granulata acclimated to salinities of 10 psu (hyperregulation) and 35 psu (osmoconformation). Sections of the hepatopancreas of adult male crabs were treated with hematoxylin-eosin; Masson trichrome; Alcian Blue (pHs 2.8, 1.0, 0.5); Toluidine Blue (pHs 5.6, 4.2); periodic acid Schiff; Sudan Black and Red. At salinity 35 psu, the hepatopancreas of both species exhibited typical histological features, whereas at salinity 10 psu, detachment of the basal lamina, desquamated epithelium, disrupted brush border, loss of intercellular cohesion, hypertrophied tubular lumen, and hemolymph infiltration between cells were observed in some zones. Resorptive cells (R-cells) and vacuoles of blister-like cells (B-cells) of both species show a higher glycogen content at 35 psu than at 10 psu. At lower salinities, the cytoplasm of the different cell types evidence higher contents of carboxylated GPs in N. granulata and of su If at ed GPs in C. angulatus. At both salinities, and at the two pHs in N. granulata and at pH 5.6 in C. angulatus, the brush border, the vacuoles of B-cells and the peritrophic membrane show metachromasia. R-cell vacuoles and the cytoplasm of all cell types -except for the E-cells-at all salinities in both species show abundant lipid droplets. The results of the present study contribute significant data to the histophysiology of crustacean decapods, favoring the comprehension of the complex adjustment mechanisms facing saline stress in euryhaline crabs.
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The objective of this study is to estimate the point-of-reserve-saturation 50 (PRS50) of stage III (JIII) and 1-gram (J1g) juveniles of Cherax quadricarinatus and to evaluate the early and late effects of feeding restriction on survival, growth and hepatopancreas structure. The experiments consisted of different feeding treatments followed by continuous starvation until molting to the following stage (restriction period). After molting, juveniles were fed daily until the end of the experiment (refeeding period). The PRS50 estimated for JIII was 2.05±0.11days, according to which 2 feeding days were required for 50% of the JIII to molt to JIV. However, the value of growth increment and the presence of hepatopancreatic abnormalities showed that these molted juveniles were not in optimal conditions. Their hepatopancreas showed a significant recovery during the refeeding period. This suggests that mortality in JIII exposed to a feeding restriction period close to the PRS50 occurs earlier than in the following stages and that the survivors recover after a refeeding period. The PRS50 of JIII could be used to test offspring quality, with the immediate advantage of reducing maintenance costs of poor-quality juveniles. The PRS50 estimated for J1g was 9.19±0.54days; those fed for less than 9days exhibited higher mortality during the restriction period, and those of F8 and F9 had histological abnormalities after the refeeding period. The mortality in J1g of F9 increased at the end of the experiment, suggesting that although they would be able to molt in a proportion similar to the control, they die later as a consequence of the restriction period. In this study, the relative wet hepatopancreas weight (RHW) was similar among treatments and between both experiments even when histological examination showed nutritional stress, implying that the RHW estimated with wet weight is a poor indicator of nutritional status. An adequate management in terms of reducing the amount of food and the use of proper tools for monitoring the health of cultured animals are essential for improving profits. In this context, the values of PRS50 and the information obtained from the present study are useful to establish a feeding schedule for the production of C. quadricarinatus.
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Severe mortalities due to luminescent vibrios occurred in pond-cultured Penaeus monodon juveniles particularly in the first 45 days of culture. Luminescent vibriosis epizootics led to reduced shrimp production due to mortalities and slow growth of affected stocks. Monitoring of bacterial population in the rearing water of several ponds was conducted from the time the ponds were flooded with water until 60 days of culture to understand the course of infection. Results showed that the occurrence of mortalities was preceded by a shift of the bacterial profile of the rearing water in infected ponds, notably the dominance of luminescent vibrios. Comparison of bacterial load in the rearing water and water source (river or open sea) showed elevated luminescent Vibrio counts in the former at 12 days to 3 weeks after initial entry of water. Histopathology of affected shrimps showed the hepatopancreas as the target organ of infection where severe inflammatory responses in the intertubular sinuses were seen.
Article
The digestive gland of crustacean is involved in various metabolic activities, including the synthesis and secretion of digestive enzymes that begin the process of food digestion, intracellular digestion and absorption of nutrients, storage of reserves, and disposal of waste products. It consists of two glandular lobes which extensively subdivide to form a complex of blind-ending tubules, whose size, surface area, and digestive cells are associated with intracellular digestion and the nutritional status of the organism. The aim of this paper was to study the morphology of the digestive gland in various lobster species and calculate the surface area of tubules, lumen and digestive cells (R-, F-, and B-cells) and their ratios to total tubule surface area. The similarity in ratios obtained in this study between individual lobsters suggests that the method developed in this study can be successfully applied to a range of species. This study describes a novel image processing algorithm for the automatic measurement of the hepatopancreas structure using stained cross sections of digestive gland tubules.The proposed new methodology could be used for studying the physiology and nutrient metabolism of lobsters and other crustaceans. The computer-aided analysis described in this paper is accurate for the quantitative assessment of the lobster’s digestive gland structure.
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This study examines the influence of feeding on digestive enzyme activities in the black tiger prawn, Penaeus monodon, using histological and biochemical techniques. Juvenile P. monodon (>50 mm total length) were sacrificed after feeding at sequential time intervals, with unfed controls also sacrificed at the same time intervals. Resin histology revealed that there were no morphological changes in the digestive gland F-cells between fed and unfed P. monodon over time. There were no significant changes in α-amylase specific activities in fed animals over time post feeding, nor were there any changes in starved animals through time. Trypsin and α-glucosidase activities were significantly higher in unfed animals than fed animals. There was a significant peak in α-glucosidase activities at 0 min post feeding but this could not be conclusively determined as an influence from feeding because of high variability and the near negligible levels of α-glucosidase activities. A peak in lipase activity was observed at 60 min post feeding, but because there was no significant differences in lipase activities between fed and unfed animals it was therefore inconclusive. Results from histological and biochemical analyses failed to prove that feeding had a significant influence on digestive enzyme production of P. monodon and suggest that digestive enzyme production in P. monodon may be continuous and is not strongly influenced by feeding.
Article
Midgut gland cells of Penaeus monodon postlarvae were investigated by electron microscopy after starvation and refeeding with different diets.Well nourished postlarvae could be starved for 5 days without irreversible detriment. They recovered easily on a good diet. Only R cells were affected by a short starvation period. After 13 days of food deprivation the structures of all cell types were disintegrated. The postlarvae were able to starve for a maximum of 15 days. The most diversified ultrastructure was obtained by refeeding with cod liver oil and 2s (mixed diet). Casein was not well utilized. Sucrose was the poorest diet. The midgut gland of Penaeus monodon seems to be lipid oriented. Only fat was used as storage material; glycogen was lacking.Different ultrastructures were induced in R cells by a given diet after longer starvation periods. The reestablishment of their ultrastructure by means of a definite diet seems to be impossible after a certain period of starvation (point of no return).R cells are very sensitive to different diets. B cells show slight reactions, while F and E cells remain relatively unchanged. This indicates that R cells could be used to monitor the nutritional value of prawn diets in aquaculture.
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This paper presents an overview of the image analysis techniques in the domain of histopathology, specifically, for the objective of automated carcinoma detection and classification. As in other biomedical imaging areas such as radiology, many computer assisted diagnosis (CAD) systems have been implemented to aid histopathologists and clinicians in cancer diagnosis and research, which have been attempted to significantly reduce the labor and subjectivity of traditional manual intervention with histology images. The task of automated histology image analysis is usually not simple due to the unique characteristics of histology imaging, including the variability in image preparation techniques, clinical interpretation protocols, and the complex structures and very large size of the images themselves. In this paper we discuss those characteristics, provide relevant background information about slide preparation and interpretation, and review the application of digital image processing techniques to the field of histology image analysis. In particular, emphasis is given to state-of-the-art image segmentation methods for feature extraction and disease classification. Four major carcinomas of cervix, prostate, breast, and lung are selected to illustrate the functions and capabilities of existing CAD systems.
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The growth in penaeid shrimp aquaculture has been mirrored by the emergence of a number of serious diseases, some of which (e.g. white spot syndrome virus - WSSV) spread rapidly across the globe through movement of infected stock. The World Organisation for Animal Health (OIE) lists six penaeid shrimp pathogens of which three are notifiable in the EU: WSSV (listed as non-exotic to the EU), Taura syndrome virus (TSV) and yellow head disease (YHD) (both listed as exotic). EU Member States (MS) must determine a status for non-exotic diseases (e.g. disease free, unknown, infected). In developing a policy for WSSV, import risk analysis (IRA) can be used to systematically assess the risks of introduction and justify risk mitigation to maintain freedom. OIE guidelines recommend that countries assess the risk of disease introduction via commodities, not listed by the OIE as safe, and apply sanitary measures if necessary. The sanitary measures necessary to maintain freedom from WSSV may not be compatible with current EU animal health legislation. The recent revision by OIE of products listed as safe for international trade strengthens the case for the risks of TSV and YHD introduction into the EU to be assessed. Freedom from WSSV is an important criterion for the development of shrimp aquaculture in the EU. However, in developing disease control policy, governments need to balance the potentially competing interests of all stakeholders, including consumers. Thus economic modelling of the impact of possible sanitary measures on consumer prices of imported products is needed to support decision making. The creation of disease free compartments and post-import risk mitigation for commodities may create the conditions conducive to the development of shrimp aquaculture whilst minimising the costs of maintaining disease freedom.
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We investigated the morphometric characteristics of the hepatopancreatic epithelium in the terrestrial isopod Porcellio scaber during acclimatization to laboratory conditions, during the daily cycle, the molt cycle, and fasting. The hepatopancreatic epithelium was analyzed using computer-assisted microscopy of serial sections of the hepatopancreatic tubes. In addition, the abundance, the distribution, and the size of lipid droplets in the hepatopancreatic epithelium were recorded. The experimental animals were collected in the field and transferred to the laboratory. The hepatopancreatic epithelium was thinner and lipid droplets reduced after 2 months of acclimatization to laboratory conditions. The daily cycle and the molt cycle affected neither the epithelial thickness nor the abundance of lipid droplets. But in animals fasted for 2 weeks, these two parameters were significantly reduced. Based on both the epithelial thickness and the abundance of lipid droplets in B cells, we propose criteria for estimating the stress status of the animals. With the possibility to determine the stress status, many studies on isopods gain in relevance.
Article
Transmission of white spot syndrome virus (WSSV) in shrimp has been reported to occur by feeding and immersion. In the present study, the impact of the molt process and artificial lesions in the cuticle on shrimp susceptibility to WSSV was examined using intramuscular and immersion routes. For the intramuscular route, Penaeus (Litopenaeus) vannamei shrimp (n=450) were injected with 10(-2.3) up to 10(2.7) shrimp infectious dose 50% end point (SID(50)) of WSSV in early and late post-molt, inter-molt, early and late pre-molt; resp. A-, B-, C-, D1- and D2-stage. The resulting infection titers demonstrated that no difference (p>0.05) in susceptibility existed between different molt stages when virus was injected. For the waterborne route, shrimp in different molt stages were immersed in seawater containing 10(4)SID(50)ml(-1) of WSSV. In a first study, P. vannamei (n=125) incubated in cell culture flasks, became infected with WSSV mostly in post-molt stages. In a second study, 2 groups of P. vannamei (n=100) and P. monodon (n=100) were transferred into plastic bags to prevent damage to the cuticle; and in 1 group a pleopod was cut off prior to incubation. Induction of damage increased infection significantly (p<0.05) in A-stage from 0-40% to 60-100%, in B-stage from 0-20% to 40-60%, in C-stage from 0-20 to 20-60%, while infection was 0% in D-stages with both immersion methods. This study proved that shrimp are more susceptible to WSSV infection via immersion after molting than in the period before molting and wounding facilitates infection.
Article
Tumours have only rarely been observed in the decapod crustaceans, a large animal group of more than 10,000 species that includes the commercially important and well investigated shrimp, lobsters, crayfish and crabs. Analysis of the literature and information from cancer and diseases data bases revealed a total of 15 incidences, some of them being questionable. Even in the long-lived species, which can reach life spans of almost 100 years, neoplasias are virtually unknown. The data published so far suggest that the strikingly different frequencies of carcinogenesis between decapods and other well investigated animal groups like mammals, fish, insects and molluscs is based on differences of the metabolic pathways for carcinogens, the immune systems, and the regulation of stem cells. Therefore, representatives of the Decapoda may serve as useful models to study how organisms can successfully prevent or control spontaneously and environmentally induced cell proliferation. A particularly promising candidate for in-depth investigation of these topics is the marbled crayfish, a rather new clonal lineage that is presently being introduced as a laboratory model in development and epigenetics.
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Monoclonal antibodies (mabs) specific for Penaeus monodon haemocytes were produced by immunising mice with membrane lysates of shrimp haemocytes. Four mabs (WSH 6, WSH 7, WSH 8 and WSH 16) were characterised using flow cytometry, light microscopy, laser scanning microscopy, electron microscopy and immunoprecipitation. WSH 6 recognised a carbohydrate determinant on an 85 kDa molecule. WSH 7, WSH 8 and WSH 16 recognised 50, 35 and 115 kDa molecules, respectively. For all mabs, differences in amount and intensity of the labelling were found when haemocytes were fixed immediately in 2% formaldehyde in Alsever's Solution (AS), compared with non-fixed haemocytes that were kept in AS (which reduced activation of the haemocytes) or in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of >80% of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mabs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS and in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different size and electron density. Immunoreactive extracellular thread-like material could be observed in cells in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and the lowest for WSH 16. Double labelling revealed that all mabs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis is put forward that immunoreactive molecules recognised by these mabs, are related to haemocyte activation factors.
Article
The aim of this study is to describe the ultrastructure of the hepatopancreas of P. argentinus in intermoult. P. argentinus hepatopancreas was studied using standard TEM techniques. Each tubule consists of four cellular types: E (embryonic), F (fibrillar), R (resorptive) and B (blister like). E-cells have embryonic features and some of them were found in mitosis. F, R and B cells possess an apical brush border. F-cells have a central or basal nucleus, a conspicuous RER, and dilated Golgi cisternae. R cells show a polar organization of organelles in three areas: apical, with numerous mitochondria and sER tubules, a central area with the nucleus and RER, and a basal area containing a sER-like tubule system and mitochondria. B-cells were observed at different stages of their life cycle. In an early differentiation stage they comprise an apical endocytotic complex and Golgi vesicles. The fusion of endocytotic and Golgi vesicles originates subapical vacuoles. During maturation, a big central vacuole is formed by coalescence of subapical vacuoles. The central vacuole is eliminated by holocrine secretion. The ultrastructure suggests that F-cells synthesize proteins, R-cells storage nutrients and B-cells have a secretory or excretory function, and confirms the independent origin of F, B and R cells from the embryonic cells.
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