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1.
Gene ; 763: 144956, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32739586

RESUMO

Sox transcription factors play essential roles in a variety of critical physiological processes. Still, members of the sox gene family have not yet been genome-wide identified in shrimps. In this study, a total of five members of the sox gene family were identified from the genome of Pacific white shrimp Litopenaeus vannamei and classified into three subgroups based on the conserved HMG-box domain. Among them, three belong to the SoxB subgroup (one in B1 and two in B2), one in the SoxC subgroup, and one in the SoxE subgroup. The five sox genes had different sex-biased expression in some tissues. Sox21, soxB1, and sox14 had a higher expression in ovary than in testis. In comparison, sox4 had a male-biased specific expression in the gonad, hepatopancreas, gill, and eyestalk. There was no difference in soxE gene expression between testis and ovary. During embryonic development, the expression level of three sox genes (soxB1, sox21, and soxE) was higher in gastrulation stage compared to previous stages, declined in limb bud stage and then increased in intramembrane nauplius stage; the expression of sox4 was detected in blastula stage and continued to increase in the following two stages and then surged in intramembrane nauplius stage; the highest expression of sox14 was in the fertilized egg stage, and the expression level decreased with the development of the embryo. These results suggest that the shrimp sox gene family may be involved in gametogenesis, tridermogenesis, and neurogenesis.


Assuntos
Proteínas de Artrópodes/genética , Penaeidae/genética , Fatores de Transcrição SOX/genética , Animais , Proteínas de Artrópodes/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Brânquias/embriologia , Brânquias/metabolismo , Hepatopâncreas/embriologia , Hepatopâncreas/metabolismo , Masculino , Especificidade de Órgãos , Ovário/embriologia , Ovário/metabolismo , Penaeidae/embriologia , Fatores de Transcrição SOX/metabolismo , Testículo/embriologia , Testículo/metabolismo
2.
Parasitol Res ; 119(9): 3013-3022, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32740752

RESUMO

Babesiosis is a tick-borne protozoonosis caused by Babesia, which can cause fever, hemolytic anemia, hemoglobinuria, and even death. Babesia microti is a parasite found in rodents and can be pathogenic to humans. In this study, the full-length cDNA of a B. microti cysteine protease (BmCYP) was expressed and the recombinant rBmCYP protein analyzed and characterized. BmCYP is encoded by an ORF of 1.3 kb, with a predicted molecular weight of 50 kDa and a theoretical pI of 8.5. The amino acid sequence of BmCYP exhibits an identity of 32.9 to 35.2% with cysteine proteases of Babesia ovis, Babesia bovis, and Theileria, respectively. The results of the proteinase assays show that rBmCYP has cysteine protease enzymatic activity. In addition, we demonstrate that tick cystatins rRhcyst-1 and rRhcyst-2 were able to effectively inhibit the activity of rBmCYP; the inhibition rates were 57.2% and 30.9%, respectively. Tick cystatins Rhcyst-1 and Rhcyst-2 were differentially expressed in ticks that fed on Babesia-infected mice relative to non-infected control ticks. Our results suggest that BmCYP is a functional enzyme with cysteine protease enzymatic activity and may be involved in tick-B. microti interactions.


Assuntos
Proteínas de Artrópodes/metabolismo , Babesia microti/enzimologia , Cistatinas/metabolismo , Cisteína Proteases/metabolismo , Proteínas de Protozoários/metabolismo , Carrapatos/metabolismo , Carrapatos/parasitologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Babesia bovis/química , Babesia bovis/enzimologia , Babesia bovis/genética , Babesia microti/química , Babesia microti/genética , Babesiose/parasitologia , Cistatinas/genética , Cisteína Proteases/química , Cisteína Proteases/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Carrapatos/genética
3.
Gene ; 756: 144914, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32574759

RESUMO

The life history of the Chinese mitten crab (Eriocheir japonica sinensis) includes two migrations: a feeding migration and a reproductive migration. Ambient salinity is one of the most critical factors during migration. In this study, the salinity adaptation mechanism of Chinese mitten crabs was simulated using continuous salinity changes. The expression of six key genes [Na+/K+-ATPase α subunit (NAK-α), V-type H+-ATPase subunit A (VHA-A), Zinc transporter (ZnT), Cl- channel protein 2 (CLCN2), ubiquitin/ribosomal S27 fusionprotein (S27), and glutathione S-transferase (GST)] and the activities of three enzymes [Na+/K+-ATPase (NAK), V-type H+-ATPase (VHA), and glutathione S-transferase (GST)] were evaluated in ten groups exposed to a range of salinity changes during mariculture based on the transcriptome data obtained from short term salinity-induced crabs (ES) compared to control group in freshwater crabs (EF). The results revealed that different genes exhibited different roles in physiological regulation. In total, 3,599 unigenes were significantly and differentially expressed in a comparison between the EF and ES treatments. A novel modulation of gene expression and the corresponding enzyme activity of NAK and VHA exhibited similar patterns. As genes related to osmoregulation, NAK and VHA showed similar patterns of both gene expression and enzyme activity in mariculture. During the gradual change in salinity from 0‰ to 25‰ and back to 0‰, the gene expression and enzyme activities of NAK and VHA initially increased (0‰ â†’ 10‰), weakened (10‰ â†’ 20‰) and then increased again (20‰ â†’ 25‰ â†’ 0‰). S27 could serve as a reference gene in the expression analysis of Chinese mitten crabs under salinity stress. ZnT and CLCN2 were involved in osmoregulation as functional proteins. Our findings provide insights into the regulation mechanisms employed during the migration of the Chinese mitten crab.


Assuntos
Braquiúros/genética , Braquiúros/fisiologia , Osmorregulação , Adaptação Fisiológica , Migração Animal , Animais , Proteínas de Artrópodes/genética , Proteínas de Transporte de Cátions/genética , Água Doce , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Brânquias/fisiologia , Masculino , Salinidade , Análise de Sequência de RNA
4.
PLoS One ; 15(5): e0232880, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32401761

RESUMO

The southern king crab (SKC) Lithodes santolla is an important commercial species in southern South America. Fishing pressure has caused the deterioration of its stocks. Currently, culture techniques are being developed for producing SKC juveniles to enhance the natural population and to recover the fishing stock. Therefore, it is necessary to know about physiology, energetic and nutritional requirements for SKC maintenance in hatchery. Thus, this study aims to evaluate the biochemical and physiological changes in the midgut gland, muscle and hemolymph of juveniles, pre-adults and adults of wild SKC. The energetic reserves, digestive enzymes activity, amino acid profile and energy were quantified in twelve juveniles, ten pre-adult, and ten adult crabs. Juveniles showed high glycogen and low lipids in the midgut gland, and low proteins and low lactate in muscle. In the hemolymph, juveniles had high lipids. Pre-adults had high glycogen and lipids in the midgut gland, and both high protein and lactate in muscle. In the hemolymph, pre-adults had high lipids. Adults had low glycogen and high lipids in midgut gland, and both high proteins and high lactate in muscle. In hemolymph, adults had high glucose and lactate. Juveniles and pre-adults had high proteinase activity, whereas adults had high lipase activity. Major essential amino acids of SKC were arginine, methionine, and tryptophan, and the non-essential amino acids were glycine, aspartic acid and glutamic acid. On another hand, SKC had similar energy in the midgut gland and muscle, regardless of the ontogenetic stage. Moreover, we demonstrated that the biochemical energy calculation underestimates the actual measured values by a calorimeter. Thus, our results help to understand the physiological changes, energetic and nutritional requirements of L. santolla, and this study is a baseline for research on diet formulation for maintaining this species under culture conditions.


Assuntos
Anomuros/fisiologia , Aquicultura/métodos , Proteínas de Artrópodes/genética , Optogenética/métodos , Aminoácidos/análise , Ração Animal , Animais , Anomuros/citologia , Anomuros/crescimento & desenvolvimento , Dieta , Metabolismo Energético , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hemolinfa , Masculino , Músculos/química
5.
Proc Natl Acad Sci U S A ; 117(21): 11399-11408, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32398368

RESUMO

Spiders are one of the most successful venomous animals, with more than 48,000 described species. Most spider venoms are dominated by cysteine-rich peptides with a diverse range of pharmacological activities. Some spider venoms contain thousands of unique peptides, but little is known about the mechanisms used to generate such complex chemical arsenals. We used an integrated transcriptomic, proteomic, and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins. Twenty-six of the 33 superfamilies are disulfide-rich peptides, and we show that 15 of these are knottins that contribute >90% of the venom proteome. NMR analyses revealed that most of these disulfide-rich peptides are structurally related and range in complexity from simple to highly elaborated knottin domains, as well as double-knot toxins, that likely evolved from a single ancestral toxin gene.


Assuntos
Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Venenos de Aranha/química , Animais , Proteínas de Artrópodes/análise , Austrália , Dípteros/efeitos dos fármacos , Dissulfetos , Evolução Molecular , Feminino , Perfilação da Expressão Gênica , Espectrometria de Massas , Peptídeos/análise , Peptídeos/química , Peptídeos/genética , Filogenia , Conformação Proteica , Proteômica/métodos , Venenos de Aranha/genética , Venenos de Aranha/toxicidade , Aranhas/genética
6.
Insect Biochem Mol Biol ; 120: 103347, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32114158

RESUMO

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites.


Assuntos
Proteínas de Artrópodes/genética , Sistemas CRISPR-Cas , Edição de Genes , Mutagênese , Oxirredutases/genética , Tetranychidae/genética , Animais , Proteínas de Artrópodes/metabolismo , Oxirredutases/metabolismo
7.
Vet Parasitol ; 279: 109064, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32143012

RESUMO

Tick serpins are involved in enzyme activity, food digestion, blood-feeding, immune response and anticoagulation. Little is known about the potential roles of serpins in tick reproduction. RHS8, a serpin from the tick Rhipicephalus haemaphysaloides, has an open reading frame 1212 bp long and encodes a protein that has 404 amino acids and a predicted molecular weight of 45 kDa. RHS8 exhibits 89.58 % amino acid identity with RmS15 in Rhipicephalus microplus. RHS8 was expressed primarily in larvae and nymphs. RHS8 mRNA expression in the ovaries, fat bodies and salivary glands were up-regulated from feeding to ovipositing ticks. RNAi results showed that RHS8 dsRNA-injected ticks had a lower body weight, longer feeding time, fewer eggs laid and lower egg hatchability. Tick reproduction, such as egg laying and hatching, was disrupted by RNAi. Compared with the control group, ovaries of the RHS8 interference group were light brown color, indicating a reduction in yolk granule accumulation. Western blot results showed that the expression of RHVg3 and RHVg4 proteins in ovaries was reduced in the RHS8 dsRNA-injected group. These results indicate that RHS8 is related to tick reproduction and its interference affects vitellogenesis.


Assuntos
Proteínas de Artrópodes/genética , Rhipicephalus/fisiologia , Serpinas/genética , Vitelogênese/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Feminino , Larva/crescimento & desenvolvimento , Larva/fisiologia , Ninfa/crescimento & desenvolvimento , Ninfa/fisiologia , Filogenia , Interferência de RNA , Rhipicephalus/genética , Rhipicephalus/crescimento & desenvolvimento , Alinhamento de Sequência , Serpinas/química , Serpinas/metabolismo
8.
Gene ; 736: 144421, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32018014

RESUMO

5-Aminolevulinic acid synthase (ALAS) is the rate-limiting enzyme in the biosynthesis of heme, a prosthetic group that is found in hemoproteins, including those involved in molting. To better understand the roles of ALAS in L. vannamei (LvALAS), we analyzed its sequence and tissue distribution, the effects of age and bacterial infection on its gene expression, and the effects of LvALAS gene silencing. We also examined the expressions of three hemoproteins, the cytochrome oxidase subunit I (COX I) and subunit IV (COX IV) and catalase. Three LvALAS splicing variants were found in the hepatopancreas, with the main splicing variant having an open reading frame that encodes 532 aa. LvALAS transcripts were found in each of the eleven tissues tested in this study, with the highest gene expression in the intestine. The transcript abundances of LvALAS, COX I and COX IV in the hepatopancreas and stomach tended to decrease with age. LvALAS and catalase gene expressions significantly increased in the stomach after V. parahaemolyticus infection. LvALAS gene expression in the hepatopancreas, stomach and intestine (12- and 24-hours post-injection) was relatively lower in dsALAS-injected shrimp than in PBS-injected shrimp. All the PBS-injected shrimp molted after 8-10 days while no molting activity was observed in the dsALAS-injected shrimp group within the 14 days post-injection period. Our results provide evidence that (1) only the housekeeping form of ALAS exists in L. vannamei; LvALAS gene expression (2) decreases with age and (3) increases after bacterial infection; and (4) an ALAS-dependent pathway is necessary for proper molting in L. vannamei.


Assuntos
5-Aminolevulinato Sintetase/genética , Proteínas de Artrópodes/genética , Expressão Gênica/genética , Penaeidae/genética , Sequência de Aminoácidos , Ácido Aminolevulínico/metabolismo , Animais , Clonagem Molecular/métodos , Hepatopâncreas/metabolismo , Hepatopâncreas/patologia , Intestinos/patologia , Penaeidae/patogenicidade , Filogenia , Alinhamento de Sequência , Estômago/patologia
9.
PLoS Negl Trop Dis ; 14(2): e0007758, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32049966

RESUMO

Amblyomma americanum ticks transmit more than a third of human tick-borne disease (TBD) agents in the United States. Tick saliva proteins are critical to success of ticks as vectors of TBD agents, and thus might serve as targets in tick antigen-based vaccines to prevent TBD infections. We describe a systems biology approach to identify, by LC-MS/MS, saliva proteins (tick = 1182, rabbit = 335) that A. americanum ticks likely inject into the host every 24 h during the first 8 days of feeding, and towards the end of feeding. Searching against entries in GenBank grouped tick and rabbit proteins into 27 and 25 functional categories. Aside from housekeeping-like proteins, majority of tick saliva proteins belong to the tick-specific (no homology to non-tick organisms: 32%), protease inhibitors (13%), proteases (8%), glycine-rich proteins (6%) and lipocalins (4%) categories. Global secretion dynamics analysis suggests that majority (74%) of proteins in this study are associated with regulating initial tick feeding functions and transmission of pathogens as they are secreted within 24-48 h of tick attachment. Comparative analysis of the A. americanum tick saliva proteome to five other tick saliva proteomes identified 284 conserved tick saliva proteins: we speculate that these regulate critical tick feeding functions and might serve as tick vaccine antigens. We discuss our findings in the context of understanding A. americanum tick feeding physiology as a means through which we can find effective targets for a vaccine against tick feeding.


Assuntos
Proteínas de Artrópodes/química , Ixodidae/fisiologia , Proteoma/química , Saliva/química , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Cromatografia Líquida , Comportamento Alimentar , Feminino , Ixodidae/química , Ixodidae/genética , Masculino , Proteoma/genética , Proteoma/metabolismo , Coelhos , Saliva/metabolismo , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismo , Espectrometria de Massas em Tandem , Infestações por Carrapato/parasitologia
10.
J Therm Biol ; 87: 102477, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32001020

RESUMO

Temperature is a critical abiotic factor that causes physiological changes in arthropods. However, little is known about the effect of heat stress on the antioxidant responses of Araneae species. Hylyphantes graminicola is a dominant predator in many cropping systems in China. In the present study, the effect of short-term heat stress (36, 38, 40 or 42 °C) on the reactive oxygen species (ROS) levels, the activities of antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], peroxidases [POD] and glutathione-S-transferases GST]), total antioxidant capacity (TAC), malondialdehyde (MDA) concentrations and survival of H. graminicola spiderlings and adults were investigated. The results showed that H. graminicola adults had a significantly higher survival rate compared to spiderlings at 40 °C. The heat stress increased ROS contents in H. graminicola. The SOD, CAT, POD and GST activities increased in spiderlings and adults under heat stress. These data suggest a defensive function for these enzymes in alleviating oxidative damage. Specifically, SOD plays a key role in reducing the high level of superoxide radicals in spiderlings and adults. Moreover, the POD and CAT capabilities for scavenging H2O2 in spiderlings were similar, and CAT may play a more important role than POD in scavenging H2O2 in adults at 42 °C. The spiderling TAC increased significantly at 40 and 42 °C, and the adult TAC was stable at 36-40 °C but decreased at 42 °C. These data suggest that TAC was insufficient in H. graminicola adults under more severe stress conditions. These results further our understanding of the physiological response of Araneae species exposed to heat stress.


Assuntos
Resposta ao Choque Térmico , Espécies Reativas de Oxigênio/metabolismo , Aranhas/metabolismo , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Catalase/genética , Catalase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , Aranhas/fisiologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
11.
Vet Parasitol ; 279: 109043, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32070900

RESUMO

Dermacentor marginatus is one of the main tick species in northwestern China, and is a vector of various tick-borne pathogens. Tick control method largely depends on chemical agents, but the disadvantages of using such approach would cause environmental damage and the risk of developing tick resistance to acaricides. Vaccination of tick protective antigen is an eco-friendly approach which is an alternative and promising method to mitigate tick infestation in livestock. In the study, a mu-class glutathione S-transferase (GST) sequence of D. marginatus was cloned and the recombinant protein (rDmGST) was expressed. Transcriptional level of the GST was measured together with native GST activity of the tick. Finally, A vaccine trial on rabbits against D. marginatus was proceeded to evaluate the anti-tick effect of rDmGST. Results reveled that the CDs of the D. margiantus glutathione S-transferase mu 1 gene has 669 base pair nucleotide sequence encoding a 223 amino acid. The deduced GST protein sequence had over 95 % similarity with that of D. variabilis. The rDmGST was efficiently expressed soluble and purified by His trap affinity chromatography. Enzyme activity of native GST and transcriptional profiles of the GST showed up-regulation in different stages and organs of D. marginaus during blood feeding. Polyclonal antibody reacted with rDmGST in Western blotting. Tick challenge on rDmGST inoculated rabbits showed reductions in adult female engorgement rate, total egg mass and egg hatching rate with an overall vaccine efficacy of 43.69 %. The results of the experiment indicated the GST has potential value to be an effective protective antigen of D. marginatus.


Assuntos
Antígenos/análise , Proteínas de Artrópodes/genética , Dermacentor/efeitos dos fármacos , Dermacentor/genética , Glutationa Transferase/genética , Controle de Ácaros e Carrapatos , Infestações por Carrapato/veterinária , Vacinas/imunologia , Animais , Proteínas de Artrópodes/metabolismo , Feminino , Glutationa Transferase/metabolismo , Coelhos , Infestações por Carrapato/prevenção & controle
12.
Parasit Vectors ; 13(1): 105, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32103780

RESUMO

BACKGROUND: Anaplasma ovis is a gram-negative, tick-borne obligate intraerythrocytic pathogen, which causes ovine anaplasmosis in small ruminants worldwide. VirB10 of A. ovis is an integral component of the Type IV Secretion System (T4SS). The T4SS is used by bacteria to transfer DNA and/or proteins undeviatingly into the host cell to increase their virulence. To more thoroughly understand the interaction between A. ovis and Dermacentor silvarum, a vector containing the virb10 gene of A. ovis was used as a bait plasmid to screen interacting proteins from the cDNA library of the D. silvarum salivary gland using the yeast two-hybrid system. METHODS: The cDNA of the D. silvarum salivary gland was cloned into the pGADT7-SmaI vector (prey plasmid) to construct the yeast two-hybrid cDNA library. The virb10 gene was cloned into the pGBKT7 vector to generate a bait plasmid. Any gene auto-activation or toxicity effects in the yeast strain Y2HGold were excluded. The screening was performed by combining the bait and prey plasmids in yeast strains to identify positive preys. The positive preys were then sequenced, and the obtained sequences were subjected to further analyses using Gene Ontology, UniProt, SMART, and STRING. Additionally, the interaction between the bait and the prey was evaluated using the glutathione S-transferase (GST) pull-down assay. RESULTS: A total of two clones were obtained from the cDNA library using the yeast two-hybrid system, and the sequence analysis showed that both clones encoded the same large tegument protein, UL36. Furthermore, the proteins GST-UL36 and His-VirB10 were successfully expressed in vitro and the interaction between the two proteins was successfully demonstrated by the GST pull-down assay. CONCLUSIONS: To our knowledge, this study is the first to screen for D. silvarum salivary gland proteins that interact with A. ovis VirB10. The resulting candidate, UL36, is a multi-functional protein. Further investigations into the functionality of UL36 should be carried out, which might help in identifying novel prevention and treatment strategies for A. ovis infection. The present study provides a base for exploring and further understanding the interactions between A. ovis and D. silvarum.


Assuntos
Anaplasma ovis/metabolismo , Proteínas de Artrópodes/metabolismo , Proteínas de Bactérias/metabolismo , Dermacentor/metabolismo , Dermacentor/microbiologia , Sistemas de Secreção Tipo IV/metabolismo , Anaplasma ovis/genética , Animais , Proteínas de Artrópodes/genética , Proteínas de Bactérias/genética , Dermacentor/genética , Interações Hospedeiro-Parasita , Ligação Proteica , Glândulas Salivares/metabolismo , Glândulas Salivares/microbiologia , Técnicas do Sistema de Duplo-Híbrido , Sistemas de Secreção Tipo IV/genética
13.
Artigo em Inglês | MEDLINE | ID: mdl-31958500

RESUMO

Elongation of very long-chain fatty acid 4 (Elovl4) proteins participate in the biosynthesis of long-chain and very long-chain polyunsaturated fatty acids (LC-PUFA and VLC-PUFA). In the present study, an elovl4 cDNA was cloned from the swimming crab Portunus trituberculatus by PCR techniques and functionally characterized using recombinant expression in yeast Saccharomyces cerevisiae. The elovl4 cDNA sequence contained an open reading frame of 1038 base pairs, encoding a protein of 346 amino acids. The elovl4 has typical Elovl structures, with transmembrane domains (6) and a histidine box. The elovl4 was expressed in various tissues analyzed, with the highest expression found in intestine and hepatopancreas, followed by stomach and eyestalk. The functional characterization of Elovl4 yeast showed that the P. trituberculatus Elovl4 can elongate C18-22 polyunsaturated fatty acids (PUFA), reaching in some cases products of C24 and C26. Along its ability to elongate PUFA, the P. trituberculatus Elovl4 was also efficient in the elongation of saturated fatty acids, with 28:0 and 30:0 being prominent elongation products. These results provide insight into the LC-PUFA biosynthetic capability of commercially important species of crustaceans.


Assuntos
Proteínas de Artrópodes/genética , Braquiúros/genética , Elongases de Ácidos Graxos/genética , Ácidos Graxos Insaturados/biossíntese , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/metabolismo , Braquiúros/enzimologia , Braquiúros/metabolismo , Clonagem Molecular , Elongases de Ácidos Graxos/metabolismo , Filogenia , Homologia de Sequência de Aminoácidos
14.
Parasitol Res ; 119(3): 773-781, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31897786

RESUMO

We report Armillifer moniliformis species infecting the endemic Sri Lankan brown palm civet (Paradoxurus montanus) from the Knuckles Range Forest Conservation Area, Sri Lanka. Larval stages of A. moniliformis were found during the postmortem of three civet cats found dead. Morphological studies were done by a light microscope and a scanning electron microscope (SEM). Histopathological examination was conducted using tissue samples obtained from the liver. For the molecular analysis, DNA was extracted from the isolated third-stage larvae. The NADH dehydrogenase subunit 5 (ND5) and the second internal transcribed spacer region (ITS-2), a portion of the large subunit nuclear ribosomal DNA (28S), a portion of 18S ribosomal rRNA gene (18S), and cytochrome c oxidase subunit 1 gene (COX1) were amplified using polymerase chain reaction (PCR). Excysted third-stage larvae were observed in the lungs, omentum, the pleural cavity, the abdominal cavity, and the surface of the spleen and the pericardium. Around 88 third-stage larvae were isolated from three civet cats. First-stage larvae in the liver were surrounded by outer fibrous layer over the inner germinal layer and filled with clear fluid. Slight hemorrhage, leukocyte infiltration, and mild hepatocellular degeneration in the liver were observed. The SEM examination indicated the unique oral apparatus comprises the oval-shaped mouth opening in between two pairs of curved, retractile hamuli. The sequences obtained for ND5, ITS-2, 28S, 18S, and COX1 were 301, 382, 325, 414, and 644 bp in length respectively. Morphology, sequence similarity search, sequence alignment, and phylogenetic analysis identified this parasite as A. moniliformis.


Assuntos
Doenças Parasitárias em Animais/parasitologia , Pentastomídeos/citologia , Pentastomídeos/genética , Viverridae/parasitologia , Animais , Proteínas de Artrópodes/genética , Larva/classificação , Larva/citologia , Larva/genética , Larva/crescimento & desenvolvimento , Doenças Parasitárias em Animais/patologia , Pentastomídeos/classificação , Pentastomídeos/crescimento & desenvolvimento , Filogenia , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico/genética , Análise de Sequência de DNA
15.
Sci Rep ; 10(1): 1287, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992795

RESUMO

The Northern spot shrimp, Pandalus platyceros, a protandric hermaphrodite of commercial importance in North America, is the primary target species for shrimp fisheries within Southeast Alaska. Fishery data obtained from the Alaska Department of Fish and Game indicate that spot shrimp populations have been declining significantly over the past 25 years. We collected spot shrimps in Southeast Alaska and measured reproductive-related morphological, gonadal and molecular changes during the entire life history. The appendix masculina, a major sexual morphological indicator, is indicative of the reproductive phase of the animal, lengthening during maturation from juvenile to the male phase and then gradually shortening throughout the transitional stages until its complete disappearance upon transformation to a female. This morphological change occurs in parallel with the degeneration of testicular tissue in the ovotestis and enhanced ovarian vitellogenesis. Moreover, we obtained the entire mRNA sequence of the yolk protein precursor, vitellogenin, and monitored its transcript levels throughout the entire shrimp life-cycle. Vitellogenin transcript levels in the hepatopancreas increased in the early transitional stage until reaching a peak prior to extruding eggs. Such transcriptomic analyses, coupled with a comprehensive description of the gonad, external sex characters and timing of the reproductive life history of spot shrimps contribute to a better understanding of the hermaphroditic reproduction process in the cold Southeast Alaskan waters. This knowledge can contribute to a revision of current conservation efforts to maintain wild populations sustainable for both commercial and ecological considerations.


Assuntos
Proteínas de Artrópodes , Pesqueiros , Pandalidae , RNA Mensageiro , Análise de Sequência de RNA , Transcriptoma , Alaska , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Conservação de Recursos Energéticos , Pandalidae/genética , Pandalidae/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
16.
Artigo em Inglês | MEDLINE | ID: mdl-31647988

RESUMO

This review discusses the reaction catalysed, and the structure and function of the cellulase, endo-ß-1,4-glucanase and the hemicellulase enzymes, ß-1,3-glucanase and endo-ß-1,4-mannase that are present in numerous invertebrate groups with a diverse range of feeding specialisations. These range from microbial deposit and filter feeders, micro and macrophagous algal feeders, omnivores to herbivorous leaf litter and wood feeders. Endo-ß-1,4-glucanase from glycosyl hydrolase family 9 (GH9) digests cellulose like ß-1,4-glucans from a range of materials. As it hydrolyses crystalline cellulose very slowly, it is a poor cellulase. Where tested, the enzyme has dual endo-ß-1,4-glucanase and lichenase activity. Its presence does not necessarily indicate the ability of an animal to digest cellulose. It only indicates the ability to digest ß-1,4-glucans and its function, which is discussed in this review, should be considered with reference to the substrates present in the diet. ß-1,3-glucanase (laminarinase) belongs to glycosyl hydrolase family 16 (GH16) and hydrolyses ß-1.3-glucans. These polysaccharides are present in the cell walls of algae, protozoans and yeast, and they also occur as storage polysaccharides within protozoans and algae. Depending on their site of expression, these enzymes may function as a digestive enzyme or may be involved in innate immunity. Enzymes present in the digestive fluids or tissues, would be digestive. Haemolymph GH16 proteins may be involved in innate immunity through the activation of the phenol oxidase system. Insect GH16 proteins expressed within the haemolymph have lost their catalytic residues and function as ß-glucan binding proteins. In contrast, crustacean GH16 proteins expressed within the same tissue, have retained the catalytic residues and thus possibly their ß-1,3-glucanase activity. The potential function of which is discussed. Endo-ß-1,4-mannase from glycosyl hydrolase family 5, subfamily 10 (GH5_10) hydrolyses mannan, glucomannan and galactomannan. These hemicelluloses are present in the cell walls of plants and algae and also function as storage polysaccharides within legume and palm seeds. They are digestive enzymes whose high expression in some species suggests they are a major contributor to hemicellulose digestion. They may also provide the animal with substantial amounts of monosaccharides for energy.


Assuntos
Proteínas de Artrópodes , Celulase , Glicosídeo Hidrolases , Invertebrados , Filogenia , Polissacarídeos/metabolismo , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Celulase/genética , Celulase/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Invertebrados/enzimologia , Invertebrados/genética
17.
Mol Genet Genomics ; 295(2): 299-311, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31724065

RESUMO

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.


Assuntos
Astacoidea/genética , Desenvolvimento Embrionário/genética , Imunidade Inata/genética , Transcriptoma/genética , Animais , Proteínas de Artrópodes/genética , Astacoidea/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Anotação de Sequência Molecular
18.
Fish Shellfish Immunol ; 96: 319-329, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31805414

RESUMO

Viral immediate early (IE) genes encode regulatory proteins that are critical for viral replication. WSV056 is an IE protein of white spot syndrome virus (WSSV), an important pathogen of farmed shrimp. It targets the host Rb protein(s) and, according to a previous study, may enhance the replication of the viral genome. However, the ectopic expression of WSV056 in transgenic Drosophila melanogaster exerted an inhibitory effect on the replication of Drosophila C virus (DCV). Transcriptome study using Affymetrix GeneChip suggested that the enrichment of serine proteases (SPs) likely accounts for DCV inhibition in WSV056-overexpressing Drosophila. Injection of recombinant WSV056 to the WSSV natural host Litopenaeus vannamei enhanced the expression of the SP family member prophenoloxidase-activating enzyme 2 (LvPPAE2) and conferred shrimp with more resistance to WSSV infection. LvPPAE2 knockdown contributed to decreased expression of antimicrobial peptides LvAlf1 and LvLyz1, reduced hemolymph phenoloxidase activity, and increased virus load, suggesting that LvPPAE2 is involved in the host defense against WSSV infection. Taken together, these results suggest that wsv056 plays a role in restricting viral replication by inducing the SP-mediated immune responses in the host.


Assuntos
Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/imunologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Análise Serial de Proteínas
19.
Fish Shellfish Immunol ; 96: 152-160, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31794843

RESUMO

C-type lectins are Ca2+-dependent carbohydrate-binding proteins containing one or more carbohydrate-recognition domains (CRDs). C-type lectins play crucial roles in innate immunity, including nonself-recognition and pathogen elimination. In the present study, two C-type lectins (designated ReCTL-1 and ReCTL-2) were identified from the shrimp Rimicaris exoculata which dwells in deep-sea hydrothermal vents. The open reading frames of ReCTL-1 and ReCTL-2 encoded polypeptides of 171 and 166 amino acids respectively, which were both composed of a signal peptide and a single CRD. The key motifs determining the carbohydrate binding specificity of ReCTL-1 and ReCTL-2 were respectively Glu-Pro-Ala (EPA) and Gln-Pro-Asn (QPN), which were firstly discovered in R. exoculata. ReCTL-1 and ReCTL-2 displayed similar pathogen-associated molecular pattern (PAMP) binding features and they bound three PAMPs-ß-glucan, lipopolysaccharide and peptidoglycan-with relatively high affinity. In addition, both could efficiently recognize and bind Gram-positive bacteria, Gram-negative bacteria and fungi. However, ReCTL-1 and ReCTL-2 exhibited different microbial agglutination activities: ReCTL-1 agglutinated Staphylococcus aureus and Saccharomyces cerevisiae, while ReCTL-2 agglutinated Micrococcus luteus, Vibrio parahaemolyticus and V. fluvialis. Both ReCTL-1 and ReCTL-2 inhibited the growth of V. fluvialis. All these results illustrated that ReCTL-1 and ReCTL-2 could function as important pattern-recognition receptors with broad nonself-recognition spectra and be involved in immune defense against invaders, but their specificities are not the same. In addition, the two ReCTLs possessed different carbohydrate binding specificities from each other and from the classical pattern: ReCTL-1 with an EPA motif bound d-galactose and l-mannose, while ReCTL-2 with a QPN motif bound d-fucose and N-acetylglucosamine.


Assuntos
Decápodes/genética , Decápodes/imunologia , Imunidade Inata/genética , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Lectinas Tipo C/química , Filogenia , Receptores de Reconhecimento de Padrão/metabolismo , Alinhamento de Sequência
20.
Fish Shellfish Immunol ; 96: 53-61, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31801694

RESUMO

Target of rapamycin (TOR) is an atypical of Ser/Thr protein kinase that plays an important role in many aspects such as cell growth, reproduction, differentiation, cell cycle regulation, autophagy and apoptosis. However, little information is known about the enzyme in crustaceans. Here, a novel TOR was identified from shrimp Penaeus vannamei (PvTOR) and its biological functions were investigated in response low temperature stress. The PvTOR gene encoded a polypeptide of 2464 amino acids with an estimated molecular mass of 279.4 kD and a predicted isoelectronic point (pI) of 7.30. Phylogenetic analysis revealed that PvTOR shared high similarity with other known species. PvTOR mRNA was detected in all the tested tissues and highest transcription in muscle and hepatopancreas. PvTOR transcriptional level was up-regulated significantly at 1.5 h and 3 h, and down-regulated at 12 h and 24 h after low temperature stress. TEM and autophagy indicator system GFP-PvLC3 suggested that low temperature induced autophagy generation. ROS, Ca2+ concentration and apoptosis rate were increased significantly in TOR-knockdown shrimp after low temperature stress. The autophagy associated gene ATG8II/I, PvBeclin-1, PvATG14, apoptosis gene PvPARP, Pvcasp-3, PvBAX and Pvp53 transcripts, and casp-3/8 activity in hemocyte were increased significantly in TOR-knockdown group shrimp at 3 h after low temperature stress. Additionally, THC counts of TOR-knockdown group were significantly higher than the dsGFP group. In summary, these results suggested that PvTOR plays an important role in the adaptation mechanisms of shrimp at low temperature by regulating autophagy and apoptosis.


Assuntos
Proteínas de Artrópodes/genética , Temperatura Baixa/efeitos adversos , Penaeidae/genética , Penaeidae/imunologia , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Animais , Apoptose/genética , Proteínas de Artrópodes/metabolismo , Autofagia/genética , Filogenia , Análise de Sequência de DNA , Estresse Fisiológico , Serina-Treonina Quinases TOR/metabolismo
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