Estudio de la calidad del agua en estanques e implementación de un protocolo de buenas prácticas acuícolas en la producción de camarón marino en Camaronera Eben Ezer, San Alejo, La Unión / Study of water quality in ponds and implementation of a Good Aquaculture Practices Protocol in the production of marine shrimp at the Camaronera Eben-Ezer, San Alejo, La Unión

La Escuela Especializada en Ingeniería ITCA FEPADE, MEGATEC La Unión, a través de la carrera Técnico en Manejo Integrado de Recursos Costero Marino con especialidad en Acuicultura y Pesquería, realizó esta investigación en asocio con Camaronera Eben Ezer. El proyecto tuvo como objetivo la caracterización de la calidad física, química y biológica del agua del Golfo de Fonseca y el Estero El Chapernal, previo al desarrollo de dos ciclos de cultivo de camarón marino Litopenaeus vannamei, durante el cultivo y antes de las descargas de agua a los efluentes receptores. La metodología se desarrolló en tres fases, de junio a diciembre de 2021. Fase de campo: toma de parámetros físicos, químicos y biológicos en 8 puntos de muestreos, Estero El Chapernal, Golfo de Fonseca y estanques de producción. Fase de laboratorio: se realizó sembrando muestras de agua en medios de cultivo para aislamiento, crecimiento e identificación de bacterias. Tercera fase: procesamiento de datos y análisis de resultados. Se identificaron tres tipos de bacterias: Psudomonas, Vibrios y Heterótrofas. En el primer ciclo de junio a agosto, éstas no reportaron crecimiento, esto pudo deberse a que la transición de la época seca a época lluviosa influyó en los factores físicos y químicos, como la temperatura y el pH. Durante el primer ciclo, la calidad del agua del Estero El Chapernal mantuvo estándares idóneos para el crecimiento del camarón. El segundo ciclo presentó un crecimiento exponencial de las bacterias Heterótrofas y Pseudomonas sobrepasando los límites de referencia establecidos en la normativa vigente. Como parte de la proyección social se efectuó transferencia de conocimientos y tecnología durante la producción, se establecieron mejoras para la operatividad y se les dotó de un protocolo de producción acuícola.

The Escuela Especializada en Ingeniería ITCA FEPADE, MEGATEC La Unión, through the major of Técnico en Manejo Integrado de Recursos Costero Marino con especialidad en Acuicultura y Pesquería, carried out this research in association with Camaronera Eben Ezer. The objective of the project was to characterize the physical, chemical and biological quality of water in Golfo de Fonseca and Estero El Chapernal, prior to the development of two culture cycles of the marine shrimp Litopenaeus vannamei, during the culture and before discharges of water to receiving effluents. The methodology was developed in three phases, from June to December 2021. Field phase: collection of physical, chemical and biological parameters at 8 demonstration points, Estero El Chapernal, Golfo de Fonseca and production ponds. Laboratory phase: it was carried out by planting water samples in culture media for isolation, growth and identification of bacteria. Third phase: data processing and analysis of results. Three types of bacteria were identified: Psudomonas, Vibrios and Heterotrophs. In the first cycle from June to August, growth was not reported, due to the fact that the transition from the dry season to the rainy season influenced in physical and chemical factors, such as temperature and pH. During the first cycle, the water quality of Estero El Chapernal maintained optimal standards for shrimp growth. The second cycle presented an exponential growth of Heterotrophic and Pseudomonas bacteria, exceeding the reference limits established in current regulations. As part of the social projection, knowledge and technology were transferred during production, they were profoundly improved for operability and they were provided with an aquaculture production protocol.

Size distribution and sex ratio between populations of the artisanal harvested land crab Cardisoma guanhumi (Decapoda: Gecarcinidae), with the estimation of relative growth and size at sexual maturity in Puerto Rico / Distribución de tallas y proporción de sexos entre poblaciones del cangrejo de tierra cosechado artesanalmente Cardisoma guanhumi (Decapoda: Gecarcinidae), con la estimación del crecimiento relativo y tamaño en la madurez sexual en Puerto Rico

Abstract Introduction: Knowledge of growth patterns, sex ratio, and sexual maturity are of importance to exploited populations. The land crab Cardisoma guanhumi is an artisanal and subsistence exploited species in Puerto Rico. However, the growth patterns and sexual maturity of the local populations are not known. Objectives: This study has a double objective: (1) to compare the size and sex structure between populations and (2) to model the relative growth of structures related to reproduction to estimate the average size of morphometric sexual maturity (MSM) for both males and females. Methods: A total of 2 849 specimens were captured from nine dispersed populations on the island between 2001 and 2020. Carapace width (CW) was measured as an estimator of the absolute size of all individuals, together with the propodus length (PL) in males and the abdomen width in females (AW). Differences in length structure between sexes and populations were tested by applying goodness-of-fit tests based on Kernel Density Estimators (KDE). The relative growth pattern was modeled adjusting a spline from which the maximum of its second derivative was calculated as an estimator of the MSM, and bootstrapping was used to generate confidence intervals. Results: Differences were found in size structures, between sexes, and between sites. Our estimates of morphometric sexual maturity resulted in a sexual maturity size for males between 57.9 and 79.0 mm CW, while in females, morphological maturity occurs between 43.8 to 51.5 mm CW. Conclusions: We found inter-population differences in body size that can be attributed to differences in the history of changes of land use and the exploitation biased towards larger individuals, though differences in recruitment should also be considered. Current regulations in Puerto Rico protect female crabs but not to larger male crabs. The regulation establishes that crabs smaller than 64 mm carapace width cannot be captured, leaving immature male crabs over 64 mm CW unprotected. We suggest considering different size limits depending on the sex of the crab. This will allow the full range of sizes where sexual maturity is reached to be protected, increasing the likelihood of the population's size to increase.

Resumen Introducción: El conocimiento de los patrones de crecimiento, la proporción de sexos y la madurez sexual son de suma importancia para las poblaciones explotadas. El cangrejo terrestre Cardisoma guanhumi es una especie explotada artesanalmente y para subsistencia en Puerto Rico. Sin embargo, se desconocen los patrones de crecimiento y la madurez sexual para la isla. Objetivos: Este estudio tiene un doble objetivo: (1) comparar el tamaño y la estructura sexual entre las poblaciones y (2) modelar el crecimiento relativo de estructuras relacionadas con la reproducción (propodo en machos y abdomen en hembras) para estimar el tamaño promedio de madurez sexual morfométrica (MSM). Métodos: Se capturaron un total de 2 849 especímenes de nueve poblaciones dispersas en la isla, entre 2001 y 2020. Se midió el ancho del caparazón (AC) como un estimador del tamaño absoluto de todos los individuos, junto con el largo de propodo en los machos y el ancho del abdomen en las hembras. Las diferencias en la estructura de tallas entre sexos y poblaciones se probaron aplicando la prueba de bondad de ajuste basado en estimaciones de densidad kernel (KDE). El patrón de crecimiento relativo se modeló ajustando un spline a partir del cual se calculó el máximo de su segunda derivada como estimador del MSM y se utilizó bootstrapping para generar intervalos de confianza. Resultados: Se encontraron diferencias en las estructuras de tallas, entre sexos y poblaciones. Nuestras estimaciones de madurez sexual morfométrica dieron como resultado un tamaño de madurez sexual para los machos entre 57.9 y 79.0 mm AC, mientras que, en las hembras, la madurez morfométrica se produce entre 43.8 y 51.5 mm AC. Conclusiones: La diferencia en el tamaño de la población se puede atribuir a las diferencias entre poblaciones en el cambio de uso de la tierra y la presión de explotación asociada con el sesgo hacia la captura de individuos más grandes o el reclutamiento. Las regulaciones actuales en Puerto Rico brindan protección a las hembras, pero no a los machos más grandes. El reglamento establece que no se pueden capturar cangrejos de menos de 64 mm de AC dejando desprotegidos los cangrejos machos inmaduros de más de 64 mm AC. Sugerimos considerar diferentes límites de tamaño según el sexo del cangrejo. Esto permitirá proteger un rango más amplio de tamaños donde se alcanza la madurez sexual, aumentando la probabilidad de un incremento en el tamaño de la población.

Crayfish hemocytes develop along the granular cell lineage.

Despite the central role of hemocytes in crustacean immunity, the process of hemocyte differentiation and maturation remains unclear. In some decapods, it has been proposed that the two main types of hemocytes, granular cells (GCs) and semigranular cells (SGCs), differentiate along separate lineages. However, our current findings challenge this model. By tracking newly produced hemocytes and transplanted cells, we demonstrate that almost all the circulating hemocytes of crayfish belong to the GC lineage. SGCs and GCs may represent hemocytes of different developmental stages rather than two types of fully differentiated cells. Hemocyte precursors produced by progenitor cells differentiate in the hematopoietic tissue (HPT) for 3 ~ 4 days. Immature hemocytes are released from HPT in the form of SGCs and take 1 ~ 3 months to mature in the circulation. GCs represent the terminal stage of development. They can survive for as long as 2 months. The changes in the expression pattern of marker genes during GC differentiation support our conclusions. Further analysis of hemocyte phagocytosis indicates the existence of functionally different subpopulations. These findings may reshape our understanding of crustacean hematopoiesis and may lead to reconsideration of the roles and relationship of circulating hemocytes.

The sustainability of rice-crayfish coculture systems: a mini review of evidence from Jianghan plain in China.

BACKGROUND: Rice-crayfish coculture (RC) uses 10% of the paddy field area for crayfish ditches, with rice cultivated in the remaining area, providing staple carbohydrates alongside fish rich in protein and essential oils. These systems rely on complex interactions, involving nutrient cycling, fish feeding, soil physicochemical modification, and insect-pest predation, and have important socio-economic effects. Past studies have considered only one or a few of these aspects pertinent to food system sustainability, and there remains a need for an integrated assessment of RC systems. In this mini-review, we collate data from two field experiments and three field surveys published across eight papers to synthesize the overall sustainability of RC systems in Jianghan Plain in China. RESULTS: In deep groundwater fields, the RC rice yield was 30-55% lower than for typical rice-rapeseed (RR) rotations, while CH4 emission and pesticide use were 41-96% and 50% lower, respectively. In shallow groundwater fields, RC rice yield was similar to typical rice monoculture (RM) cultivation, while CH4 emission and pesticide use were 28-41% and 17% lower for RC cultivation. Field survey data indicated that RC can improve soil nutrient and organic matter content significantly compared with RM cultivation, and also increase the diversity of fauna and flora in the paddy field whilst reducing the incidence of chilo suppressalis (a major rice pest). Feed inputs to RC systems increased crayfish yield by 31-71% and reduced the fraction of N inputs lost to the environment from 71% to 41%. CONCLUSION: We conclude that RC systems with feed inputs in areas with shallow groundwater can deliver improved food security, sustainability, and resilience through ecological intensification. © 2020 Society of Chemical Industry.

Áreas en riesgo de invasión por Procambarus clarkii (Decapoda: Cambaridae) un cangrejo de río introducido en Colombia / Areas at risk of invasion by Procambarus clarkii (Decapoda: Cambaridae) a crayfish introduced in Colombia

Introducción: El proceso de invasión biológica es una de las mayores amenazas a la biodiversidad y ecosistemas, con actuales y potenciales impactos en la salud pública y conservación. Procambarus clarkii es un crustáceo decápodo, originario del sur de Estados Unidos y noreste de México, que puede adaptarse a diferentes condiciones ambientales debido a su plasticidad ecológica. Objetivo: Se caracterizó el nicho ecológico fundamental existente de Procambarus clarkii con el fin de predecir las áreas con idoneidad ambiental para el potencial establecimiento de la especie en Suramérica y Colombia. Métodos: Usamos modelos de nichos ecológicos calibrados en el área nativa, elaborados con el algoritmo Maxent, basados en datos de presencia extraídos de GBIF y variables hidroclimáticas de ecosistemas acuáticos a una resolución de 1 km2. Resultados: En Suramérica el modelo indicó un potencial geográfico amplio de invasión mostrando áreas ambientalmente idóneas para la presencia y expansión hacia Colombia, Venezuela, Perú, Ecuador, Brasil, Guyana, Surinam, Bolivia, Uruguay, Argentina, Paraguay y Chile. En Colombia, el modelo predijo que las áreas idóneas se ubican principalmente en el norte y oriente, incluidos diversos ecosistemas, como: bosques tropicales, bosques basales, bosques riparios y sabanas. La especie tiene una alta posibilidad de expandirse hacia áreas de distribución de latitudes bajas, ocupando zonas hacia la parte norte de la región Caribe colombiana, en departamentos de Magdalena, Cesar, Córdoba y Atlántico. También se predijo áreas con idoneidad ambiental en el oriente de Colombia, hacia la extensión de la planicie oriental de la Orinoquia, una región de baja altitud en: Arauca, Casanare, Meta y Vichada. Conclusión: En este estudio se aplican modelos de nichos ecológicos, que puede ser de interés en la planeación de estrategias o la creación de planes de manejo, como sistemas de alerta temprana para evitar el establecimiento de esta especie.

Introduction: Biological invasions are a major threat to biodiversity and ecosystems, with current and potential impacts on public health and conservation. Procambarus clarkii is a decapod crustacean, native to the Southern United States and Northeastern Mexico, which may adapt to different environmental conditions due to its ecological plasticity. Objective: The existing fundamental ecological niche of Procambarus clarkii was characterized in order to predict areas with environmental suitability for the potential establishment of the species in South America and Colombia. Methods: We used models of calibrated ecological niches in the native area, elaborated with the Maxent algorithm, based on occurrence data extracted from GBIF and hydroclimatic variables of aquatic ecosystems at a resolution of 1 km2. Results: The model indicated a wide geographic area for invasion potential in South America, predicting environmentally suitable areas for the presence and expansion towards Colombia, Venezuela, Peru, Ecuador, Brazil, Guyana, Surinam, Bolivia, Uruguay, Argentina, Paraguay and Chile. In Colombia, suitable areas predicted by the model are located mainly in the North and East of the country, spanning a diversity of ecosystems, such as tropical forests, basal forests, riparian forests and savannas. The species has a high possibility of expanding into low latitude distribution areas, occupying areas towards the Northern part of the Colombian Caribbean region, in the departments of Magdalena, Cesar, Cordoba and Atlántico. Suitability areas environmental were also predicted in Eastern Colombia, towards the extension of the Eastern plain of the Orinoquia, a low altitude region in Arauca, Casanare, Meta and Vichada. Conclusion: This study applies ecological niche models, which may be of interest in the planning of strategies or the creation of management plans, such as early warning systems that prevent the establishment of this species.

Recycling biofloc waste as novel protein source for crayfish with special reference to crayfish nutritional standards and growth trajectory.

Screening of novel feedstuffs, that too for data-deficient (nutritionally) animals, is somewhat ambiguous or problematic. Through systematic meta-analyses, the present study formulated most up-to-date crayfish nutritional standards, against which a recyclable waste (biofloc biomass, BM) from intensive aquaculture systems was assessed as a novel protein source. Growth trajectory dependencies and thermal growth coefficient qualifying for good growth in crayfish (TGC 0.5-0.64 units) were benchmarked. Using these standards and a 7-week growth trial, BM's suitability as a novel protein source for red swamp crayfish Procambarus clarkii was evaluated through its graded inclusions in a commercial feed. Results suggest that BM can elevate growth at 33-66% inclusion in existing feed formulations. Beyond 66% inclusion, BM can deteriorate growth in crayfish due to high ash content (exceeding physiological limit > 14%), arginine deficiency (~ 14-20% lower than an optimum requirement), and insufficient non-protein energy: protein ratio (3.7 cal mg-1). Arginine is perhaps the most critical amino acid in dietary protein for crayfish, and deficient in BM. Although no critical bioaccumulation levels of heavy metals were breached by feeding 100% BM to crayfish, a mineral and heavy metal (Hg) stress seemed plausible. Crayfish raised solely on biofloc may not realize full growth potential.

The feeding of dietary Codonopsis pilosula polysaccharide enhances the immune responses, the expression of immune-related genes and the growth performance of red swamp crayfish (Procambarus clarkii).

Polysaccharides have many functions in aquatic animals and are widely used as immunopotentiators. However, despite the emergence of serious diseases, few studies have explored the effects of Codonopsis pilosula polysaccharide (CPP) on crustaceans. We studied the effects of CPP on the growth performance, nonspecific immunity, antioxidant activity and disease resistance of red swamp crayfish (Procambarus clarkii). Healthy crayfish (5.80 ± 0.1 g) were fed diets supplemented with 0% (control), 0.05%, 0.1%, 0.15%, 0.20%, and 0.30% CPP for 8 weeks. At the end of the 8-week feeding trial, the optimal final body weight (FBW), weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) were observed in the crayfish fed the diets with 0.15% and 0.20% CPP, followed by those fed the diet with 0.30% CPP and then those fed the diet with 0.10% CPP, whereas the values of these parameters were obtained with the control crayfish (P < 0.05). The crayfish fed the diets with 0.15% and 0.20% CPP exhibited a significantly higher total hemocyte count (THC) and significantly increased phenoloxidase (PO), lysozyme (LZM), hemocyte (Hc), acid phosphatase (ACP) and alkaline phosphatase (AKP) compared with those belonging to the other groups (P < 0.05). The crayfish fed the diets with 0.15% and 0.2% CPP exhibited significantly higher total superoxide dismutase (T-SOD) and glutathione peroxidase (GPx) activities, a significantly increased total antioxidant capacity (T-AOC) and a significantly lower malondialdehyde (MDA) content compared with the other groups (P < 0.05), which indicated that antioxidant capacity was significantly induced by the CPP-supplemented diets. Significantly upregulated expression of immune-related genes (anti-lipopolysaccharide factors (alf), peroxiredoxin (prx5), cathepsin B (ctsb), mitochondrial manganese superoxide dismutase (mtMnsod), cyclophilin A (cypa), glutathione peroxidase (gpx), Toll-like receptor 3 (tlr3), and heat shock protein 70 (hsp70)) was detected in the crayfish fed the diets supplemented with 0.15% and 0.20% CPP diet compared with the levels observed in the control crayfish. These results showed that dietary CPP supplementation greatly improved the growth, immunity and antioxidant capacities of crayfish, and according to the observed results, 0.15%-0.2% is the recommended optimal level of CPP dietary supplementation for crayfish.

Effects of hesperidin on the growth performance, antioxidant capacity, immune responses and disease resistance of red swamp crayfish (Procambarus clarkii).

We evaluated the effects of hesperidin on the nonspecific immunity, antioxidant capacity and growth performance of red swamp crayfish (Procambarus clarkii). A total of 900 healthy crayfish were randomly divided into six groups: the control group (fed the basal diet) and the HES25, HES50, HES75, HES100 and HES150 groups, which were fed the basal diet supplemented with 25, 50, 75, 100 and 150 mg kg-1 hesperidin, respectively. The feeding experiment lasted 8 weeks. The results indicated that compared with the control group, the crayfish groups supplemented with 50-150 mg kg-1 hesperidin had a decreased feed conversion ratio (FCR) and increased final body weight (FBW), specific growth rate (SGR) and weight gain (WG) (P < 0.05). The protein carbonyl content (PCC), reactive oxygen species (ROS) level and malondialdehyde (MDA) level in the hepatopancreas and hemocytes were significantly lower, while the total antioxidant capacity (T-AOC), glutathione peroxidase (GPx) activity, and superoxide dismutase (SOD) activity were significantly higher in the crayfish groups supplemented with 50-150 mg kg-1 hesperidin than in the control group. Supplementation with 50-150 mg kg-1 hesperidin significantly increased the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), and phenoloxidase (PO) compared with the control group (P < 0.05); upregulated the mRNA expression of cyclophilin A (CypA), extracellular copper-zinc superoxide dismutase (ecCuZnSOD), GPxs, crustin, astacidin, Toll3 and heat shock protein 70 (HSP70) (P < 0.05); and decreased crayfish mortality following white spot syndrome virus (WSSV) infection. These findings indicate that dietary hesperidin supplementation at an optimum dose of 50-150 mg kg-1 may effectively improve nonspecific immunity, antioxidant capacity and growth performance in crayfish.

Effects of metazachlor and its major metabolite metazachlor OA on early life stages of marbled crayfish.

The effects of the herbicide metazachlor and its major metabolite metazachlor OA at two concentrations, including environmentally relevant concentrations of metazachlor (0.0115 µmol/l and 0.0790 µmol/l) and metazachlor OA (0.0117 µmol/l and 0.0805 µmol/l), respectively, were evaluated on early ontogeny, growth, behaviour, oxidative stress, antioxidant enzyme levels, histology, and mortality of marbled crayfish Procambarus virginalis. Both tested concentrations of metazachlor and metazachlor OA were associated with significantly lower growth and delayed ontogenetic development compared to controls. Exposure of metazachlor at 0.0115 µmol/l and metazachlor OA at 0.0117 µmol/l and 0.0805 µmol/l resulted in significantly lower activity of total superoxide dismutase (SOD), catalase (CAT), glutathione s-transferase (GST), glutathione reductase (GR), and reduced glutathione (GSH) compared with control and resulted in gill anomalies ranging from wall thinning to focal disintegration of branchial structure. Metazachlor at the environmentally relevant concentration of 0.0790 µmol/l was associated with significant alterations of crayfish distance moved and walking speed. The potential risk associated with metazachlor use in agriculture related to effects on non-target aquatic organisms.

A comparative transcriptomic analysis in late embryogenesis of the red claw crayfish Cherax quadricarinatus.

The red claw crayfish (Cherax quadricarinatus) is an emerging and important commercial species in several countries, and is also a potential biological model in crustacean biology. However, its molecular embryonic development mechanism remains largely unknown because of a lack of genomic resources and systematic research. A comprehensive and integrated transcriptomic analysis is necessary to reveal the cell biological function, gene expression profiles, and embryo patterning that occur during embryogenesis. In the present study, transcriptomic profiles of C. quadricarinatus embryos during three developmental stages were investigated by high-throughput Illumina sequencing technology, and the genes related to development were further analyzed. In total, 49,436 unigenes were assembled and clustered, in which 13,727 were annotated in the Nonredundant database, 5087 were classified based on Gene Ontology annotations, and 2735 were associated with 189 Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, gene expression differences among the embryos stages were analyzed, and 6658 differentially expressed genes (DEGs) were identified. In total, 3300, 5211, and 1262 DEGs were identified between the eye pigments forming stage (EP) and prepare-hatching stage (PH), EP and larvae (L), as well as PH and L; meanwhile, 1595, 2540 and 680 DEGs were annotated, respectively. The fundamental developmental genes related to apoptosis, neurogenesis, and segmentation, as well as signaling pathways related to Hedgehog, MAPK, Wnt, TGF-ß and Notch, showed higher expression during the EP stage than in other two stages, indicating that the EP stage has more active biological processes than the latter stages. This transcriptome studies gene expression at different stages of embryonic development and the datasets provide a basis for understanding crustacean developmental biology and guiding seedling production.

Effect of dietary Antarctic krill Euphausia superba on the growth performance and nonspecific immunity of red swamp crayfish Procambarus clarkia.

This study aims to investigate the effects of replacing different proportions of fishmeal with Antarctic krill (AK) on the growth performance, body composition and nonspecific immunity index of red swamp crayfish Procambarus clarkia. AK was used to replace 0 (control), 25%, 50% and 100% of the fishmeal in the basic diet of crayfish to formulate four test feeds with basically equivalent nitrogen and lipid contents; these feeds were denoted AK0, AK25, AK50 and AK100, respectively. Compared with the control group, crayfish fed diets with AK replacement showed increased body weight gain; feed efficiency; survival rate; body protein content; phenoloxidase, superoxide dismutase and glutathione peroxidase activities; total haemocyte counts; number of hyaline, semigranular and granular cells; and disease resistance against Aeromonas hydrophila. Conversely, the body lipid level of these crayfish decreased relative to that of the control. However, a high AK level (AK100) does not show improvements in efficiency compared with a moderate AK level (AK50). Based on the efficiency of AK in enhancing the growth performance and nonspecific immunity of crayfish, the optimum replacement proportion of fishmeal with AK was 50%. These results confirm that AK can promote the growth of crayfish and improve their disease resistance.

Do hydroelectric dams affect the abundance and population structure of freshwater shrimps?

Freshwater shrimps play an important role in many ecological processes since they are epibenthic detritivores but also prey on other invertebrates and are predated by fishes. The knowledge about their biology allow the development of management strategies to improve the use of natural resources by avoiding overfishing and enhancing productivity. Here we evaluated the population structure of the shrimp Macrobrachium amazonicum in the river Rio Grande, upstream of the Água Vermelha hydroelectric dam. They were captured monthly from October 2017 until March 2018, in six sites along the river. The first site was ∿1000 m distant from the dam and the sixth was ∿4000 m distant. A sac-like sieve and traps were used to capture the shrimps. The sieve was dragged three times for 180o. The traps were left by the river margins for six hours. In the laboratory, the shrimps were sexed and measured (carapace length; CL). In total, 6455 individuals were captured: 4499 females (294 breeding), 1445 males, and 217 juveniles. Female mean size was 7.50 ± 3.47 mm, male mean size was 7.44 ± 2.40 mm, and juvenile mean size was 3.24 ± 1.10 mm. The smallest individual was found in site III and it was a juvenile of 1.08 mm. The largest had 20 mm and was found in site II. The smallest breeding female had 3.8 mm CL. The largest individuals were more abundant in the sites near the dam, whereas the smallest were more abundant in the farthest sites. The highest abundance was seen in sites III and IV, and the lowest, in site VI. Unlike males and juveniles, females (both breeding and non-breeding) were more abundant (Anova, p < 0.01) near the dam. By knowing the population structure of M. amazonicum it is possible to understand how they are being affected by the environmental changes caused by the hydroelectric dam

Genomic organization of the molt-inhibiting hormone gene in the red swamp crayfish Procambarus clarkii and characterization of single-nucleotide polymorphisms associated with growth.

Molt-inhibiting hormone (MIH), a neuropeptide synthesized in the eyestalk in crustaceans, is mainly responsible for the molting by negatively controlling the ecdysteroids secretion. Although there are several reports of the isolation and protein sequencing of MIH in the red swamp crayfish, little is known about the nucleotide sequence and gene organization of this neuropeptide, even less about the association of MIH polymorphisms and growth traits. Here, a 1237 bp full-length MIH cDNA was obtained from the crayfish eyestalk, which encodes a putative protein of 106 amino acids, with a 191 bp 5'-UTR and a 728 bp 3'-UTR. The MIH genomic DNA sequence is 4205 bp in length, which includes three exons interrupted by two introns, and a 929 bp 5'-flanking region. Potential transcription initiation site and transcription factor binding sites were identified in the 5'-flanking region, implying a potential role in transcriptional regulation. Seventeen SNPs in the 5'-flanking region and 3'-UTR were identified, and the associations between these SNPs and growth traits were evaluated with a two-stage design. A SNPs g. -12C > G that showed a significant association with body weight was identified. Individuals with GG genotype had a significantly higher body weight than those with CC genotype (43.98 ±â€¯9.82 g vs. 34.27 ±â€¯6.87 g; P ﹤ 0.001), indicating a beneficial effect of the G allele on the growth of red swamp crayfish. The obtained MIH gene, as well as the identified SNPs, may serve as targets for molecular marker-aided selection in growth improvement of the red swamp crayfish in future studies.

Molecular Characterization and Functional Study of Insulin-Like Androgenic Gland Hormone Gene in the Red Swamp Crayfish, Procambarus clarkii.

The androgenic gland (AG) is a male-specific endocrine organ that controls the primary and secondary sexual characteristics in male crustaceans. More evidence indicates that the insulin-like androgenic gland hormone gene (IAG) is the key male sexual differentiation factor, particularly the application of RNA interference (RNAi) technology on IAG. In this study, the full-length cDNA of IAG (termed PcIAG) was isolated from the red swamp crayfish, Procambarusclarkii. Tissue distribution analysis showed that in addition to its expression in the AG of male P. clarkii, PcIAG was widely expressed in female tissues and other male tissues. The PcIAG protein was detected in the reproductive and nervous systems of adult male P. clarkii. Additionally, RNAi results showed that the PcIAG expression could be silenced efficiently, and the male sperm maturation and release possibly present a transient adverse interference at lower doses (0.1 µg/g and 1 µg/g) of PcIAG-dsRNA (PcIAG double-stranded RNA). Dramatically, the expression level of PcIAG increased sharply shortly after the injection of higher doses (5 µg/g and 10 µg/g) of PcIAG-dsRNA, which might accelerate the maturation and release of sperm. Moreover, the expression of PcSxl (P. clarkii Sex-lethal) was detected by Quantitative Real-Time PCR (qPCR) after the injection of PcIAG-dsRNA to explore whether the PcIAG gene regulates the PcSxl gene, and we found that the PcIAG did not directly regulate PcSxl in P. clarkii. The study could help accelerate the progress of PcIAG functional research and provide a useful reference for the single-sex selective breeding of P. clarkii.

Exploring potential proteins associated with cyclin B 3'UTR in Procambarus clarkii oocytes.

Cyclin B is a ubiquitous regulatory molecule and has been implicated in mitosis and meiosis in oocytes. Phenomenon that differ in the length of cyclin B 3'UTR in crustacean has attracted much attention, although molecular details are poorly understood. The study of 3'UTR-interacting proteins could yield much information in translational regulation and the mRNA localization process. Previous studies on crayfish suggested that the 3'UTR (1300 bp) probably contains the potential regulatory sequence/motifs such as CPEs and K-box et al. In present study, using pull-down assay coupled with mass spectrometry approach allowing us to explore the potential proteins associated with the 3'UTR. We finally identified four candidate proteins including Hspg 2, Vtg, eef1a and Tuba1a, which annotated as significant roles involved in cell differentiation, lipid transporter activity, and meiotic cell cycle process. The preliminary results will contribute to the advance in understanding the translational activation of cyclin B in oocyte maturation regulation in crustacean.

The growth performance and nonspecific immunity of red swamp crayfish Procambarus clarkia affected by dietary Rhodiola rosea polysaccharide.

This study aims to investigate the effects of Rhodiola rosea polysaccharide (RRP) on the growth performance and nonspecific immunity of red swamp crayfish Procambarus clarkia. RRP was prepared by hot water extraction and partly characterised by high-performance liquid chromatography and sugar composition analyses. Three diets supplemented with three different levels of RRP (0.2, 0.6 and 1 g kg diet-1) were formulated and tested for growth performance and nonspecific immunity of red swamp crayfish Procambarus clarkii, while a diet without any RRP supplementation served as control. After 8 weeks of feeding, body weight gain, feed efficiency, survival rate, phenoloxidase activity, superoxide dismutase activity, glutathione peroxidase level, total haemocyte count and number of hyaline cells, semigranular cells and granular cells and resistance to Aeromonas hydrophila were higher than those of the control. Moreover, based on the efficiency of RRP on the growth performance and nonspecific immunity of crayfish, the optimum dose of RRP was found to be 0.6 g kg diet-1. Hence, intake of diets containing RRP could enhance the growth performance, immune responses and improve resistance of crayfish to infection by A. hydrophila.

Bt Proteins Exacerbate Negative Growth Effects in Juvenile Rusty (F. rusticus) Crayfish Fed Corn Diet.

The adoption of genetically modified (GM) crops has occurred rapidly in the United States. The transfer of GM corn byproducts from agricultural fields to nearby streams after harvest is significant and occurs well into the post-harvest year. These corn leaves, stems, and cobs then become a detrital food source for organisms, such as shredders in the stream ecosystem. Considering that the nontarget effects of Bt corn have been observed in some terrestrial organisms, we assessed whether Bt toxins affect an important aquatic organism, juvenile F. rusticus crayfish. Juvenile crayfish were fed six distinct diet treatments: two varieties of Bt corn, two non-Bt controls of herbicide tolerant corn, and two controls: fish gelatin and river detritus. Juveniles were fed these diets while housed in flow-through artificial streams that received natural stream water from a local source. Specific growth rate and survivorship of the crayfish were measured throughout the study. Juveniles fed corn diets grew significantly less and had reduced survival compared with juveniles fed fish gelatin or river detritus diets. Furthermore, juveniles fed one Bt variety of corn (VT Triple Pro®) exhibited significantly less growth than those fed one of the herbicide tolerant varieties (Roundup Ready 2®). Our study shows that corn inputs to streams may be detrimental to the growth and survivorship of juvenile crayfish and that certain Bt varieties may exacerbate these negative effects. These effects on crayfish will have repercussions for the entire ecosystem, because crayfish are conduits of energy between many trophic levels.

Antioxidant capacity and prebiotic effects of Gracilaria neoagaro oligosaccharides prepared by agarase hydrolysis.

Gracilaria is a genus of red algae widely cultivated in Asia and is notable for its economic importance as food ingredients. Neoagaro oligosaccharides (NAOSs) are products of Gracilaria that have excellent water solubility, antioxidant activity and prebiotic effect. In this study, Gracilaria crude polysaccharide was treated with agarase and hydrolyzed into NAOSs with different degrees of polymerization (DP). The compositions of the hydrolyzed NAOSs were analyzed by electrospray ionization-time of flight-mass spectrometry and thin layer chromatography. The antioxidant capacity and prebiotic effects of NAOSs with different DPs were investigated and the results showed that DP could affect the antioxidant capacity of NAOSs. The prebiotic effects of NAOSs with different DP were evaluated based on the influence on the growth of four intestinal bacteria. NAOSs promoted the growth of Lactobacillus delbrueckii subsp. bulgaricus and Sterptococcus thermophilus. The protective effect of Gracilaria NAOSs in simulated gastrointestinal juice was also studied. Finally, NAOSs with best prebiotic effects were used in Procambarus feeding experiment and exhibited promotion effect on Procambarus growth. The present study showed that Gracilaria NAOSs can be utilized as antioxidant and prebiotic additive, which had a considerable potential in food and feed industry.

Environmentally relevant concentrations of methamphetamine and sertraline modify the behavior and life history traits of an aquatic invertebrate.

Pharmaceutically active compounds are major contaminants of aquatic environments that show direct and indirect effects on aquatic organisms even at low concentrations. The aim of this study was to assess the effects of the illicit drug methamphetamine and the antidepressant sertraline on clonal marbled crayfish Procambarus virginalis. Crayfish exposed to the environmentally relevant concentrations of methamphetamine of ∼1 µg L-1 did not exhibit significant differences from unexposed controls in distance moved, velocity, and activity level with or without available shelter. Sertraline-exposed (∼1 µg L-1) crayfish were significantly more active, regardless of available shelter, and moved greater distances when shelter was available, compared to control crayfish. Crayfish exposed to methamphetamine and sertraline spent significantly more time outside the shelters compared to controls. Sertraline-exposed crayfish spawned more frequently and showed higher mortality than controls. The results suggest that the low environmental concentrations of the tested compounds could alter the behavior and life history traits of crayfish, resulting in higher reproductive effort and mortality.

Directed differentiation of granular cells from crayfish hematopoietic tissue cells.

Hemocytes are the major immune cells of crustaceans. New hemocyte production is required throughout the life cycle of these animals to maintain a functional immune system. The mechanism of crustacean hematopoiesis has just begun to be understood and new methods are needed for the investigation of this process. Here we report the directed differentiation of granular cells (GCs) from the hematopoietic tissue (HPT) cells of Cherax quadricarinatus in vitro. We started by providing the cultured HPT cells with different additives to induce possible differentiation. We found that crayfish muscle extract greatly promoted the physical status of the cells and induced the formation of refractile cytoplasmic granules. The transcription of marker genes and the production of functional prophenoloxidase further confirmed the formation of mature GCs. In our experiments, young GCs usually started to develop in ∼2 weeks post induction and over 60% of the cells became mature within 3-4 weeks. This is the first time that the fully differentiation of crustacean hemocytes is accomplished in vitro. It provides a powerful tool for in-depth study of crustacean hematopoiesis.