Agricultural drought assessment in dry zones of Tolima, Colombia, using an approach based on water balance and vegetation water stress.

Soil water balance is an essential element to consider for the management of droughts and agricultural land use. It is important to evaluate the water consumption of a crop in each of its phenological phases and the status of water reserves during critical hydrologic periods. This study developed an agricultural drought index (Standardized Soil Moisture Deficit Index - SMODI) conceptualized with a water balance model considering the vegetation stress caused by soil moisture deficit. This contribution was based on meteorological information, soil moisture from satellite images, hydrophysical properties of the soil and crop evapotranspiration. Information from 61 weather stations located in the dry zone of Tolima was used for estimating the water balance. SMODI was compared with the most common drought indexes: Standardized Precipitation - Evapotranspiration Index (SPEI), the Palmer Self-Calibrated Drought Index (scPDSI), and other eleven macroclimatic indexes. Pearson's correlation coefficients (r), Tukey's test, and analysis of variance were applied to analyze the degree of association between SMODI and the contrasting indexes on a quarterly basis. SMODI considers factors influencing soil moisture distribution and retention and the water stress thresholds that plants have evolved to withstand during drought periods. Consequently, this integrated approach enhances the assessment of agricultural drought by relying on pertinent physical processes. SMODI identified extremely dry, severe, moderate and normal drought 5 %, 3 %, 20 % and 72 % respectively conditions in areas characterized by Entisols, Inceptisols, and Andisols, where rice and fruit crops and pasturelands are cultivated. The SMODI has a good correlation with macroclimatic indexes (0.70 < r < 0.74).

Staphylococcus haemolyticus and Providencia vermicola Infections Occurring in Farmed Tilapia: Two Potentially Emerging Pathogens.

This work aimed to determine the presence of bacterial pathogens in fish with a clinical picture suggestive of infectious disease in Nile tilapia reared in Chiapas, Mexico. Blood and viscera samples were taken from healthy and diseased animals from commercial farms. Clinical and pathological examinations of each individual were performed and samples were collected for bacteriological studies. The bacterial isolates were identified and characterized by culture, biochemical tests, antibiogram, challenge tests and 16S rRNA sequencing. Staphylococcus haemolyticus and Providencia vermicola were isolated from various diseased organisms. The clinical picture caused by Staphylococcus haemolyticus was characterized by appetite disorders, neurological signs, nodulation or ulceration in different areas and congestion or enlargement of internal organs. Providenciosis in juvenile specimens caused a characteristic picture of hemorrhagic septicemia. Challenge tests performed in healthy organisms revealed that both infections caused higher mortality rates in fish (p < 0.05) compared with non-infected specimens, with 100% survival. There was 100% mortality for animals infected with P. vermicola after three days post infection and 45% for those infected with S. haemolyticus. The isolation and identification of two pathogens involved in an infection process were achieved and cataloged as potential causal agents of disease outbreaks in tilapia farming in Mexico. This is the first report of possible bacterial infection caused by S. haemolyticus and P. vermicola in tilapia farms, which are two uncommon but potentially emerging pathogens for the species.

Where are the Penaeids crustins?

Crustins are antimicrobial peptides and members of the four-disulfide core (4-DSC) domain-containing proteins superfamily. To date, crustins have only been reported in crustaceans and possess a structural signature characterized by a single 4-DSC domain and one cysteine-rich region. The high-throughput sequencing technologies have produced vastly valuable genomic information that sometimes dilutes information about previously sequenced molecules. This study aimed (1) to corroborate the loss of valuable descriptive information regarding crustin identification when high throughput sequencing carries out automatic annotation processes and (2) to detect possible crustin sequences reported in Penaeids to attempt a list considering structural similarities, which allows the establishment of phylogenetic relationships based on molecular characteristics. All crustins sequences reported in Penaeids and registered in the databases were obtained. The first list was made with the proteins reported as crustin or carcinin, excluding those that did not meet the structural characteristics. Subsequently, using local alignments, sequences were sought with high similarity even if they had been reported with a different name of crustin but with a probability of being crustin. This broader list, including proteins with high structural similarity, can help establish phylogenetic relationships of shrimp genes and the evolutionary trajectory of this antimicrobial distributed exclusively among crustaceans. Results revealed that in most sequences obtained by Sanger or transcriptomics, which met the structural criteria, the identification was correctly established as crustin. Contrarily, the sequences corresponding to crustins obtained by whole genome sequencing projects were incorrectly classified or not characterized, being momentarily "buried" in the information generated. In addition, the sequences that complied with the criteria of crustin tended to be grouped into species separated by geographical regions; for example, the crustins of the inhabitant shrimp of the American coasts differ from those corresponding to the natives of the Asian coasts. Finally, the results suggest the convenience of annotations considering the previous but correct information, even if such information was generated with previous technologies.

p53 knock-down and hypoxia affects glutathione peroxidase 4 antioxidant response in hepatopancreas of the white shrimp Litopenaeus vannamei.

Hypoxia (<2 mg O2/L) is one of the main environmental stressors that affects aquatic organisms, including the white shrimp (Litopenaeus vannamei). During hypoxia, reactive oxygen species (ROS) accumulation induces oxidative stress and damage to biomolecules. Redox state and ROS overproduction are modulated by the antioxidant system that is composed of several antioxidant enzymes, proteins, and other small compounds. Glutathione peroxidase 4 (GPx4) has emerged as an important antioxidant enzyme with cytoprotective roles. In vertebrates, antioxidant and pro-oxidant stress responses are regulated by several factors, including the p53 protein. However, little is known about GPx4 responses in crustaceans and the regulation by p53. Herein we analyzed and characterized the L. vannamei GPx4 and evaluated the responses to hypoxia and p53 knock-down. We found a unique GPx4 gene that produces five transcript variants (TVs) and only two protein isoforms with distinct cellular localization. GPx4 expression in hepatopancreas during hypoxia and p53 knock-down changed during short and long-term hypoxia, suggesting that GPx4 may be a sensitive indicator of antioxidant imbalance during stress. Knock-down of p53 induced a reduction in GPx4 expression, indicating that p53 modulates GPx4 responses during stress. This agrees with our findings of putative consensus sequences for p53 in the GPx4 gene promoter by in silico analysis. Also, the antioxidant response was effective in preventing major protein damage during hypoxia since no changes were detected in carbonylated proteins content in hepatopancreas during hypoxia. Conversely, p53 knock-down produced significant changes in carbonylated proteins.

Granulomatous bacterial diseases in fish: An overview of the host's immune response.

Bacterial diseases represent the main impediment to the development of fish aquaculture. Granulomatous diseases caused by bacteria lead to fish culture losses by high mortality rates and slow growth. Bacteria belonging to genera Streptococcus spp., Mycobacterium sp., Nocardia sp., Francisella sp., and Staphylococcus sp. have been implicated in the development of granulomatous processes. The granuloma formation and the fish's immune response continue to be the subject of scientific research. In fish, the first defense line is constituted by non-specific humoral factors through growth-inhibiting substances such as transferrin and antiproteases, or lytic effectors as lysozyme and antimicrobial peptides, and linking with non-specific phagocyte responses. If the first line is breached, fish produce antibody constituents for a specific humoral defense inhibiting bacterial adherence, as well as the mobilization of non-phagocytic host cells and counteracting toxins from bacteria. However, bacteria causing granulomatous diseases can be persistent microorganisms, difficult to eliminate that can cause chronic diseases, even using some immune system components to survive. Understanding the infectious process leading to granulomatosis and how the host's immune system responds against granulomatous diseases is crucial to know more about fish immunology and develop strategies to overcome granulomatous diseases.

Proteomic profiling of the white shrimp Litopenaeus vannamei (Boone, 1931) hemocytes infected with white spot syndrome virus reveals the induction of allergy-related proteins.

To elucidate the proteomic responses of shrimp hemocytes to white spot syndrome virus (WSSV) infection at the proteome level, a quantitative shotgun proteomic analysis was performed to detect differentially synthesized proteins in infected hemocytes of white shrimp (Litopenaeus vannamei). We identified 1528 proteins associated to 203 gene ontology (GO) categories. The most representative GO categories were regulation of cellular processes, organic substance metabolic processes and nitrogen compound metabolic processes. Most of the 83 detected up-regulated proteins are involved in DNA regulation and organization and cell signaling. In contrast, most of the 40 down-regulated proteins were related to immune defense processes, protein folding, and development. Differentially induced proteins were further analyzed at the transcript level by RT-qPCR to validate the results. This work provides new insights into the alterations of L. vannamei hemocytes at the protein level at 12 h post-infection with WSSV. Interestingly, several of the up-regulated proteins are allergy-related proteins in humans. Based on our results, we suggest a deeper analysis of the effects of this interaction on the regulation of allergy related-proteins as their up-regulation during WSSV could represent a threat to human health.

The endemic copepod Calanus pacificus californicus as a potential vector of white spot syndrome virus.

The susceptibility of the endemic copepod Calanus pacificus californicus to white spot syndrome virus (WSSV) was established by the temporal analysis of WSSV VP28 transcripts by quantitative real-time PCR (qRT-PCR). The copepods were collected from a shrimp pond located in Bahia de Kino Sonora, Mexico, and challenged per os with WSSV by a virus-phytoplankton adhesion route. Samples were collected at 0, 24, 48 and 84 h postinoculation (hpi). The VP28 transcripts were not detected at early stages (0 and 24 hpi); however, some transcript accumulation was observed at 48 hpi and gradually increased until 84 hpi. Thus, these results clearly show that the copepod C. pacificus californicus is susceptible to WSSV infection and that it may be a potential vector for the dispersal of WSSV. However, further studies are still needed to correlate the epidemiological outbreaks of WSSV with the presence of copepods in shrimp ponds.

Specific diversity of the entomopathogenic fungi Beauveria and Metarhizium in Mexican agricultural soils.

Prior knowledge of the local population structure of entomopathogenic fungi is considered an important requisite when developing microbial control strategies against major pests of crops such as white grubs. An extensive survey in the estate of Guanajuato, one of the main agricultural regions of Mexico, was carried out to determine the abundance and diversity of entomopathogenic fungi in soil. Soil collected from 11 locations was baited for entomopathogenic fungi using Galleria mellonella. In addition, all isolates were morphologically identified and selected isolates of Beauveria and Metarhizium isolates identified using Bloc and ITS or Elongation Factor 1-α and ITS sequence information respectively. Genotypic diversity was then studied using microsatellite genotyping. The proportion of isolates belonging to each genus varied amongst all locations. The species Beauveria bassiana, B. pseudobassiana and Metarhizium robertsii were found, with B. bassiana being the most abundant and widely distributed. Microsatellite genotyping showed that the 36 B. bassiana isolates were grouped in 29 unique haplotypes, but with no separation according to geographical origin.

Evaluation of the productive and physiological responses of Litopenaeus vannamei infected with WSSV and fed diets enriched with Dunaliella sp.

An experimental investigation was performed to evaluate the productive and physiological responses of Litopenaeus vannamei that were infected with WSSV and fed diets enriched with Dunaliella sp., which had a high ß-carotene content induced by nitrogen reduction in the culture medium. A basal diet containing 35% crude protein and experimental diets T1 and T2, which included 1% and 2% microalgae meal, respectively, were evaluated. Positive (infected juveniles) and negative (non-infected) controls were also evaluated. Survival was significantly higher (80%) in the two treatment groups compared to the positive control group (56%). In the negative control group, survival was 100%. Some variation was recorded for hemolymph metabolites among treatments and at distinct times post-infection; although the tendencies were not clear, some metabolites (glucose and triglycerides) appeared to decrease on the last days of the trial, probably due to their use as energy for the shrimp to thrive despite the infection. The results of the study suggest a positive effect of the dietary inclusion of Dunaliella meal on shrimp survival and an unclear effect on hemolymph metabolites.

Prevalence of viral pathogens WSSV and IHHNV in wild organisms at the Pacific Coast of Mexico.

This study investigated whether white spot syndrome virus and Infectious hypodermal and hematopoietic necrosis virus, can survive in wild invertebrates and vertebrates in the environment surrounding shrimp farms along the Pacific coast of Mexico. The evidences imply that both viruses have a potential of persisting in crabs, blue, white and brown shrimps. The most prevalent virus, IHHNV was present in 19.5% (344/1736) followed by WSSV in 3.6% (65/1736). Coinfection of WSSV and IHHNV was also detected in crabs, blue and white shrimps. This is the first prevalence report of WSSV and IHHNV associated with wildlife species in Mexico.

Effect of ante-mortem hypoxia on the physicochemical and functional properties of white shrimp (Litopenaeus vannamei) muscle stored on ice.

The effect of ante-mortem hypoxia on the physicochemical and functional properties of raw and cooked white shrimp was studied. Hue angle was greater (p ≤ 0.05) for stressed raw shrimp compared to control (greener color); whereas a lower angle was detected for cooked stressed shrimp (redder/orange coloration). In addition, hue angle increased (p ≤ 0.05) over the ice storage period for control and stressed shrimp (raw and/or cooked). Muscle hardness and shear force showed no differences when comparing control and stressed shrimp (raw and/or cooked). However, during ice storage, shear force increased (p ≤ 0.05) by 22% and 9% for control and stressed raw shrimp, respectively; in contrast, shear force and muscle hardness decreased for cooked shrimp (p ≤ 0.05). Control showed more (p ≤ 0.05) elasticity than stressed cooked shrimp. Stressed raw shrimp showed a water holding capacity 10.8% lower (p ≤ 0.05) than control. However, during the storage, water holding capacity increased (p ≤ 0.05) reaching similar values to control after day 4. Muscle protein solubility of stressed shrimp was 31% lower than control; however, no differences (p > 0.05) were observed after the second day. The thermal stability of myosin (T max) showed differences (p ≤ 0.05) among control and stressed shrimp, whereas no differences for ΔH were observed. Results showed the influence of ante-mortem hypoxia on the physicochemical and functional properties of white shrimp muscle.

Experimental evidence of metabolic disturbance in the white shrimp Penaeus vannamei induced by the Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV).

The Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) is a single-stranded DNA virus that infects several penaeid shrimp species, provoking economic losses in farmed shrimp populations estimated at several million of dollars. Furthermore, IHHNV has historically been considered an important threat for wild shrimp populations, but its real measurable impact remains unknown. Currently no treatments are available against IHHNV, and research to develop potential antiviral strategies depends on a detailed understanding of the viral life cycle. However, the exact pathophysiological events underlying the development of metabolic changes in IHHNV-infected shrimp are still unknown. Thus, the biochemical changes caused by the IHHNV infection in plasma and hepatopancreas of the economically important shrimp species Penaeus vannamei were evaluated. Glucose, lactate, total protein, glycogen, triacylglycerides, cholesterol, and total lipids were measured in healthy and IHHNV-infected shrimp. Significant changes were observed in energy substrates (glucose, lactate, triacylglycerides and cholesterol), in plasma and hepatopancreas. These changes may indicate a temporal sequestration of the host-cell metabolic pathways by the virus to maximize its replication and propagation.

Experimental infection and detection of necrotizing hepatopancreatitis bacterium in the American lobster Homarus americanus.

Necrotizing hepatopancreatitis bacterium (NHPB) is an obligated intracellular bacteria causing severe hepatopancreatic damages and mass mortalities in penaeid shrimp. The worldwide distribution of penaeid shrimp as alien species threatens the life cycle of other crustacean species. The aim of the experiment was to evaluate the possibility of experimentally infecting the American lobster (Homarus americanus) with NHPB extracted from shrimp hepatopancreas. Homogenates from infected shrimp were fed by force to lobsters. Other group of lobsters was fed with homogenates of NHPB-free hepatopancreas. After the 15th day from initial inoculation, the presence of NHPB was detected by polymerase chain reaction in feces and hepatopancreas from lobsters inoculated with infected homogenates. Necrotized spots were observed in the surface of lobster hepatopancreas. In contrast, lobsters fed on NHPB-free homogenates resulted negative for NHPB. Evidence suggests the plasticity of NHPB which can infect crustacean from different species and inhabiting diverse latitudes. Considering the results, the American lobster could be a good candidate to maintain available NHPB in vivo.

An inducible nitric oxide synthase (NOS) is expressed in hemocytes of the spiny lobster Panulirus argus: cloning, characterization and expression analysis.

Nitric oxide (NO) is a free radical gas involved in a variety of physiological processes in invertebrates, such as neuromodulation, muscle contraction and host defense. Surprisingly, little is known about the involvement of NO synthase (NOS) in the immune system of crustaceans. This work is focused on the study of the NOS gene of the spiny lobster Panulirus argus, a crustacean with commercial interest, and its relationship with the immune response to a microbial elicitor. A NOS full-length DNA was isolated from hemocytes by reverse transcription-polymerase chain reaction (RT-PCR) using degenerated primers. The open reading frame (ORF) encodes a protein of 1200 amino acids, with an estimated molecular mass of 135.9 kDa, which contains the conserved domains and binding motifs of NOS found in a variety of organisms. NOS gene expression in lobster gills, heart, stomach, digestive gland, abdominal muscle, gut and hemocytes was studied by Real Time quantitative PCR (Real Time qPCR). The expression was higher in hemocytes, heart and gills. In addition, when lobster hemocytes were exposed in vitro to Escherichia coli O55:B5 lipopolysaccharide (LPS), an increase in the NOS activity and also in the NOS gene expression evaluated by Real Time qPCR was observed, thus demonstrating the presence of an inducible crustacean NOS by a microbial elicitor of the immune response. The information is relevant in providing basic knowledge for further studies of crustacean defense mechanisms.

[Clinical outcome of a bilateral tibial pylon fracture treated with a minimally invasive technique]. / Resultado clínico de la fractura de pilón tibial bilateral tratada con técnica mínimamente invasiva.

INTRODUCTION: Intraarticular fractures of the distal tibia are traumas accounting for 1% of lower extremity fractures. The tibial pylon fracture is a metaphyseal lesion that spreads inside the ankle and is difficult to treat with any method. CASE REPORT: This is the clinical outcome at one year, of a 40-year-old male patient with a bilateral tibial pylon fracture, treated with the MIPO (minimally-invasive plate osteosynthesis) technique. RESULTS: It was possible to reduce complications such as bone and soft tissue infections, early arthrosis and pseudoarthrosis, and to achieve the patient's prompt return to activities of daily living, thus reducing the costs to the patient and the institution. DISCUSSION: Tibial pylon fractures are not frequent, let alone bilateral ones. They represent a big challenge to both the orthopedic surgeon and the patient, mainly due to the risk of injury to the skin cover. The MIPO technique modified the natural history of this type of lesion in the patient reported herein.

Role of fumonisin B1 on the immune system, histopathology, and muscle proteins of white shrimp (Litopenaeus vannamei).

White shrimps, Litopenaeus vannamei, were tested in two indoor trials to determine the effect of fumonisin B1 on (i) immune response, (ii) histopathology, and, (iii) muscle proteins. Trial 1: (0, 0.5, 0.75 and 1.0µg/g of FB1 levels, 18-day duration; shrimp 5-6g) to evaluate the FB1 effect on the immune system and histopathology response. Trial 2: (0.0, 0.5, 0.75 and 1.0µg/g of FB1 levels, 16-day duration; shrimp 5-6g) to detect FB1 effect on muscle proteins. Prophenoloxidase activity was affected by all FB1 concentrations tested. Both, total haemocyte count and phenoloxidase activity decreased by the 18th day in shrimp exposed to FB1. Marked histological changes in the hepatopancreas of shrimp fed on diet containing FB1, at the all FB1 levels tested, as well as a necrotic tissue were observed. Changes in both, electrophoretic patterns and thermodynamic properties of myosin extracted from shrimp exposed to FB1 were also observed.

Transcriptome analysis of gills from the white shrimp Litopenaeus vannamei infected with White Spot Syndrome Virus.

We report the analysis of 872 cDNA clones from a WSSV-infected white shrimp Litopenaeus vannamei gill cDNA library. Comparison against the GenBank protein and nucleotide sequences identified 87% (E < or = 10(-2)) as previously known genes, while 13% are novel sequences. The 601 ESTs (87%) represent transcripts of 276 genes. These genes were categorized into 12 groups according to their functions. The more abundant categories were (1) ribosomal proteins (21%), (2) WSSV transcripts and sequences without homology to proteins deposited in the non-redundant database (15%), (3) hypothetical proteins (12%) which include genes never described in shrimp and (4) metabolism related proteins (9%). We also found genes involved in stress and immune response; and only one involved in ion transport. Full-length sequences of keratinocyte associated protein 2 (KCP2), selenoprotein M (SelM), chicadae, prohibitin and oncoprotein nm23 are reported. Their mRNAs steady state levels in addition to ferritin, changed at different times post-WSSV infection as estimated by RT-PCR. These results suggest that WSSV alters gene expression in gills and has led to the identification of novel white shrimp specific genes.

Usage of energy reserves in crustaceans during starvation: status and future directions.

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.

The cytosolic manganese superoxide dismutase from the shrimp Litopenaeus vannamei: molecular cloning and expression.

Manganese containing superoxide dismutase (SOD) is normally a nuclear-encoded mitochondrial enzyme in eukaryotic organisms; however, a cytoplasmic manganese SOD (cMnSOD) was found in crustaceans that use hemocyanin as oxygen carrier. The complete cDNA and deduced amino acid sequence of a cMnSOD from Litopenaeus vannamei were determined. The coding sequence predicts a 287 residues protein with a unique 61 amino acids extension at the N-terminus and lacking a mitochondrial-targeting sequence. Phylogenetic analysis clusters cMnSODs and mitochondrial MnSODs in two separate groups. cMnSOD transcripts were detected in hemocytes, heart, hepatopancreas, intestine, nervous system, muscle, pleopods and gills. Since hemocytes are key defense cells and their reactions produce superoxide radicals, the infection by white spot syndrome virus on the cMnSOD transcript levels were investigated and found to increase transiently 1h post-infection and then decrease as the viral infection progressed to levels significantly lower than uninfected controls by 12h post-infection.

cDNA cloning of the lysozyme of the white shrimp Penaeus vannamei.

Lysozyme, an antibacterial protein, has been implicated in innate immunity in invertebrates, but its activity in shrimp remained to be determined. We cloned the white shrimp lysozyme cDNA using a PCR strategy and detected its activity in haemocytes using a lytic-zone assay against Micrococcus luteus. The cloning was based on a reported EST (dbEST BE18831). The deduced amino acid sequence resulted in 150 amino with 46% identity to hen egg white lysozyme. RT-PCR was used to detect lysozyme mRNA in haemocytes. Analysis of the amino acid sequence of the shrimp lysozyme showed that it belongs to the C-type family of lysozymes. Furthermore, the lysozyme amino acid sequence contained extra residues at its C-terminus, which are characteristic of marine invertebrates. This information will be useful in future studies on the molecular mechanisms of immunity in marine invertebrates.