A standardized protocol for measuring phenoloxidase in captive and wild Murray crayfish Euastacus armatus.

Innate immunity in arthropods is achieved largely through melanization which is in turn the result of the prophenoloxidase (ProPO) activation cascade; a series of biochemical reactions triggered by the immune identification of pathogen-recognition proteins (PRPs). Within this activation cascade, inactive proPO is cleaved to form the reactive enzyme phenoloxidase (PO). Methods of detecting PO are used to assess an arthropod's ability to respond to immune challenges. These detection assays have been described for some arthropods, especially those of commercial value, but none are available for Euastacus, a genus within the superfamily Parastacoidea. This study is the first step in developing a standardized protocol for the detection and quantification of PO activity in wild or captive Murray crayfish Euastacus armatus. Hemolymph extracts from 49 crayfish were assessed for PO activity using an assay measuring the conversion of l-dopa (3,4-dihydroxy-l-phenylalanine) into dopachrome. Short periods (up to 15 min) out of water did not cause any measurable change in PO activity. Phenoloxidase activity was detected in captive (n = 24, stressed) and wild (n = 25, healthy) crayfish with captive crayfish showing lower levels of PO possibly indicating immunosuppression. The proven protocol is the first of its kind to propose a standardized methodology for the detection and quantification of PO activity in Murray crayfish hemolymph as a means of determining stress.

Chitinase involved in immune regulation by mediated the toll pathway of crustacea Procambarus clarkii.

Chitinase can degrade chitin and play an essential role in animal immunity and plant defense. The immune functions of Chitinase in Procambarus clarkii (P. clarkii) remain to elucidate. Here, we identified PcChitinase 2 gene sequence from P. clarkii and studied its spatial and temporal expression profiles. The PcChitinase 2 transcribed unequally in different tissues; however, its expression was highest in those of stomach, gut, and hepatopancreas. The challenge with lipolysaccharide or peptidoglycan significantly up-regulated the expression of PcChitinase 2 in hepatopancreas. The knockdown of the PcChitinase 2 gene by double-stranded RNA suppressed most of the Toll-pathway-related immune genes (phospholipase, lectin, sptazle Cactus, serine proteikinase, anti-lipopolysaccharide factor, and Toll) production were significantly increased. Our results suggest PcChitinase 2 may be involved in the innate immune responses of P. clarkii by modulating the toll pathway.

Roles of a small GTPase Sar1 in ecdysteroid signaling and immune response of red swamp crayfish Procambarus clarkii.

Secretion-associated and ras-related protein 1 (Sar1) is a small GTPase that plays an important role in the transport of protein coated with coat protein complex II vesicles. However, its alternative roles in the biological processes of Procambarus clarkii remain unclear. Here, a sar1 gene (named as Pc-sar1) with an open reading frame of 582 bp from P. clarkii was identified. Pc-sar1 was expressed in all examined tissues with highest expression levels in muscle, which was determined by real-time PCR and western blotting. After the induction of lipopolysaccharide (LPS) and polycytidylic acid (Poly I: C), the transcriptional levels of Pc-sar1 differed in hepatopancreas, gill, muscle and intestine. In contrast, the expression of Pc-sar1 was upregulated by 20-hydroxyecdysone in these four tissues. In addition, the RNA interference of Pc-sar1 significantly affected the expression levels of immune and hormone-related genes. These results indicate that Pc-sar1 is involved in the innate immune response and ecdysteroid signaling pathway.

Using environmental DNA methods to survey for rare groundwater fauna: Detection of an endangered endemic cave crayfish in northern Alabama.

The conservation and management of subterranean biodiversity is hindered by a lack of knowledge on the true distributions for many species, e.g., the Wallacean shortfall. In recent years, several studies have demonstrated the potential of environmental DNA (eDNA) as an effective approach to detect and monitor biodiversity, including rare, threatened, and endangered taxa. However, there are few eDNA studies of groundwater fauna. Here we report the results of the development and implementation of an eDNA assay targeting a short fragment of the mitochondrial CO1 locus of a critically imperiled cave crayfish, the Sweet Home Alabama Cave Crayfish (Cambarus speleocoopi), known from just four cave systems in the Interior Plateau karst region of northern Alabama. We detected C. speleocoopi DNA from water samples collected at 5 of 16 sites sampled (caves and springs), including two historical sites as well as three additional and potentially new sites in Marshall County, Alabama. All three of these sites were within 2 km of historical sites. Our study is the first to detect a groundwater crustacean in the Interior Plateau karst region. Additionally, our study contributes to the growing literature that eDNA is a viable complementary tool for detection and monitoring of a fauna that is difficult to survey and study using traditional approaches.

Transglutaminase 1 and 2 are localized in different blood cells in the freshwater crayfish Pacifastacus leniusculus.

In the present study we show that hemocytes in the freshwater crayfish Pacifastacus leniusculus express two different transglutaminases. We describe the sequence of a previously unknown TGase (Pl_TGase1) and named this as Pl_TGase2 and compared this sequence with similar sequences from other crustaceans. The catalytic core domain is similar to the previously described TGase in P. leniusculus, but Pl_TGase2 has significant differences in the N-terminal and C-terminal domains. Further, we show conclusive evidences that these different transglutaminases are specific for different hemocyte types so that Pl_TGase1 is expressed in the hematopoietic tissue and in the cytoplasm of semigranular hemocytes, while Pl_TGase2 is expressed in vesicles in the granular hemocytes. By in situ hybridization we show that both Pl_TGase1 and Pl_TGase2 mRNA are present only in a subset of the respective hemocyte population. This observation indicates that there may be different subtypes of semigranular as well as granular hemocytes which may have different specific functions.

Dietary Bacillus amyloliquefaciens enhance survival of white spot syndrome virus infected crayfish.

Bacillus amyloliquefaciens, which is closely related to Bacillus subtilis, produces a series of metabolites that can inhibit the growth of fungi and bacteria. Here, we investigated the effect of B. amyloliquefaciens used as a probiotic on the innate immunity of the crayfish Procambarus clarkii when challenged with white spot syndrome virus (WSSV). Dietary B. amyloliquefaciens supplement significantly reduced the mortality of WSSV-challenged crayfish and reduced copy numbers of WSSV. The quantitative reverse transcription-polymerase chain reaction results showed that B. amyloliquefaciens supplement increased the expression of several immune-related genes, including Toll-like receptor, NF-κB and C-type-lectin. Further analysis showed that B. amyloliquefaciens supplement also had an effect on three immune parameters, including total hemocyte count, phenoloxidase activity and superoxide dismutase activity. In both infected and uninfected crayfish, B. amyloliquefaciens supplement significantly decreased hemocyte apoptosis. Our results showed that B. amyloliquefaciens can regulate innate immunity of crayfish and reduce the mortality following WSSV challenge. This study provides a novel insight into the potential for therapeutic or prophylactic intervention with B. amyloliquefaciens to regulate crayfish immunity and protect against WSSV infection, and also provides a theoretical basis for the use of probiotics as aquatic feed additives.

Cloning and characterization of chst11 from Procambarus clarkii involved in the host immune response of white spot syndrome virus and Aeromonas hydrophila.

Carbohydrate sulfotransferases 11 (chst11) is one of the enzymes that synthesize chondroitin sulfate (CS), which has extensive immune functions in vitro and plays a critical role in mediating the infection of host by pathogenic microorganisms. However, whether it has immune functions in crayfish is still poorly understood. In our previous study of transcriptome, chst11 was differentially expressed in susceptible individuals and resistant individuals of Procambarus clarkii after white spot syndrome virus (WSSV) injection. Thus, in this study, the sequence of chst11 was obtained from P. clarkii for the first time and analyzed, and the expression pattern of chst11 was investigated. Besides, the purified recombinant protein of chst11 effect in protection in WSSV infection was explored. The full length of chst11 was 1536 bp with an 831-bp open reading frame (ORF), which encoding 276 amino acids residues with a calculated molecular mass of 33.1 kDa. The chst11 contains a Sulfotransfer_2 domain, one N-glycosylation site and three O-glycosylation sites. Phylogenetic analysis results showed that chst11 had the highest similarity to Penaeus vannamei (79.93%). The expression pattern of chst11 in different tissues indicated that chst11 was expressed highest in gut, gill and hypodermis, lowest in testicular duct, periesophageal nerve and hemocytes. The chst11 had different expression patterns in different tissues when the crayfish was challenged by WSSV, Aeromonas hydrophila and CpG ODN. Recombinant chst11 protein significantly reduced the amount of WSSV copy number in hepatopancreas at 6 h and 12 h post injection compared to the control group injected with bovine serum albumin (BSA). It was found that chst11 protein enhanced the expression of peroxinectin, proPO in hepatopancreas and midgut and the C-type lectin (ctl) in hemocytes and hepatopancreas. Intramuscularly injection of juvenile crayfish with chst11 protein decreased 60% mortality compared to the control group with BSA. This study is the first report on the antiviral function of chst11 in the immune system of crustacean.

The N-terminal peptide generated after activation of prophenoloxidase affects crayfish hematopoiesis.

The circulating hemocytes of invertebrates are important mediators of immunity, and hemocyte homeostasis is of high importance for survival and health of crustaceans. The prophenoloxidase (proPO)-activating system is one of the most essential immune reactions, which can be activated by pattern recognition proteins from microorganisms. Activation of proPO by the proPO activating enzyme generates an N-terminal peptide, with cleavage site after Arg176, as well as the active enzyme phenoloxidase, which is the key enzyme for melanization. In the present study we demonstrate a role for the N-terminal proPO-peptide in hematopoiesis. Injection of this proPO-peptide increased the number of circulating hemocytes and especially granular hemocytes. We also show that the reactive oxygen species (ROS) production in the anterior proliferative center was enhanced after proPO peptide injection, which is a prerequisite for rapid hemocyte release from the hematopoietic tissue. Moreover, this peptide had an effect on ROS production in in vitro cultured hematopoietic cells and induced spreading of these cells within 72 h. Taken together, our findings show a role of the N-terminal proPO peptide in stimulation of hematopoiesis in crayfish, Pacifastacus leniusculus.

Acute toxicity of cypermethrin on the juvenile of red claw crayfish Cherax quadricarinatus.

In order to assess the toxicity of Cypermethrin (CYP), the 50% lethal concentration (LC50) of CYP on the juvenile of Cherax quadricarinatus is assessed. Meanwhile, the transcription level and the content in the antioxidant and biotransformation enzymes in hepatopancreas and immune enzymes in the serum of C. quadricarinatus exposed to CYP (0.1, 1, 10 and 100 ng·L-1) for 96 h were analyzed to reveal the CYP toxicity and detoxification mechanism. 24, 48, 72, 96 h LC50 were 1305.14, 424.52, 287.10 and 215.99 ng·L-1, respectively. There was no significant change of the content of enzymes at low concentration (0.16 ng·L-1). The fast increase of SOD and CAT content was observed at early stage (24 h), subsequent decreased at later stage of trail at medium concentration (0.32 and 0.63 ng·L-1). However, high concentration (1.25 ng·L-1) of CYP significantly inhibited SOD and CAT content. There was a significant increase in the level of MDA, PC and the content of GPx, EROD, CarE, GST at medium and high concentration after 72 h and 96 h exposure. The Na+-K+-ATPase, PO, ALK content decreased at medium and high concentration, especially at the 72-h and the 96-h exposure. The transcription was altered similarly to enzyme content, but the transcriptional response was generally more immediate than enzymatic response. Heat shock protein (hsp70) and multidrug resistance-associated protein 2 (abcc2) genes were up-regulated.

Cloning and Analysis of the Multiple Transcriptomes of Serine Protease Homologs in Crayfish (Procambarus clarkii).

Five different serine protease homologs (SPH) transcripts presumably or possibly resulting from alternative splicing were cloned from the hemocytes of crayfish (Procambarus clarkii) in this paper. Although different deletions of cDNA of SPH-2 and SPH-4 were found in the 5' untranslated regions, they shared the same open reading frame and encoded a 424 amino acids protein with a calculated molecular weight of 45.84 kDa compared with SPH-5. The predicted cutting site of the signal peptide was located between Ala22 and Glu23; a clip domain and a trypsin-like serine protease domain were located in the N-terminal and the C-terminal, respectively. Large deletions were found in the SPH-1 and SPH-3. Both of them lacked the clip domain. The 22 amino acids signal peptide existed in the SPH-1 coding protein, and a low complexity region (LCR) was formed in the N-terminal of it. The deduced protein of SPH-1 contained 358 amino acids with a molecular weight of 38.80 kDa. There was only one trypsin-like serine protease domain found in the C-terminal of the SPH-3 coding protein. The deduced protein of SPH-3 contained 250 amino acids with a molecular weight of 26.90 kDa. The amino acid Ser (S) of the catalytic triad in trypsin-like serine protease domain of the proteins analyzed in this paper was replaced by Gly (G), suggesting that the SPH-1, SPH-2, SPH-3, SPH-4, and SPH-5 were serine protease homologs.

Transglutaminase inhibition stimulates hematopoiesis and reduces aggressive behavior of crayfish, Pacifastacus leniusculus.

Transglutaminase (TGase) is a Ca2+-dependent cross-linking enzyme, which has both enzymatic and nonenzymatic properties. TGase is involved in several cellular activities, including adhesion, migration, survival, apoptosis, and extracellular matrix (ECM) organization. In this study, we focused on the role of the TGase enzyme in controlling hematopoiesis in the crayfish, Pacifastacus leniusculus We hypothesized that a high TGase activity could mediate an interaction of progenitor cells with the ECM to maintain cells in an undifferentiated stage in the hematopoietic tissue (HPT). We found here that the reversible inhibitor cystamine decreases the enzymatic activity of TGase from crayfish HPT, as well as from guinea pig, in a concentration-dependent manner. Cystamine injection decreased TGase activity in HPT without affecting production of reactive oxygen species. Moreover, the decrease in TGase activity in the HPT increased the number of circulating hemocytes. Interestingly the cystamine-mediated TGase inhibition reduced aggressive behavior and movement in crayfish. In conclusion, we show that cystamine-mediated TGase inhibition directly releases HPT progenitor cells from the HPT into the peripheral circulation in the hemolymph and strongly reduces aggressive behavior in crayfish.

Characterization of a cold-active transglutaminase from a crayfish, Pacifastacus leniusculus.

Transglutaminase (TGase) from signal crayfish (Pacifastacus leniusculus) and its activity at low temperatures was studied. TGase is an abundant protein in the hematopoietic (HPT) cells and this tissue was used for TGase enzyme preparation. The optimal temperature and pH for the activity of crayfish TGase were determined. We found that TGase activity at 4 °C showed nearly the same activity as at a temperature of 22 °C. TGase activity from crayfish was compared with guinea pig liver TGase activity at 4 °C and the crayfish TGase displayed a higher activity while guinea pig liver TGase had a very low activity at this low temperature. By comparing kinetic parameters to guinea pig liver TGase, the results showed that a high activity of crayfish TGase was due to a decreasing Km value for pentylamine as a substrate, while it did not affect the kcat value (at 22 °C). The amino acid sequences of a krill and a crayfish TGase, which both are cold adapted, do not give any clue to why these two enzymes are cold-adapted. These results demonstrate that crayfish TGase is adapted to have significant activity at low temperatures and since crayfish are living in quite cold waters this is an interesting adaptation of this enzyme.

Response of phase I and II detoxification enzymes, glutathione, metallothionein and acetylcholine esterase to mercury and dimethoate in signal crayfish (Pacifastacus leniusculus).

Metals and pesticides are common pollutants and the modulation of biomarkers can indicate sub-lethal influences on the physiology of organisms inhabiting impacted aquatic systems. We examined the effects of mercury and the organophosphate pesticide dimethoate on EROD, MROD, glutathione S-transferase (GST), acetylcholine esterase (AChE), metallothionein (MT) and glutathione (GSH) in the signal crayfish (Pacifastacus leniusculus). Crayfish were injected with mercury chloride or dimethoate (0.3, 0.6, 0.9 µg kg-1) and dissected after 72 h. EROD activity in the hepatopancreas did not change in response to mercury chloride treatment but exhibited a dose dependent decrease at all concentrations of dimethoate tested. MROD (hepatopancreas) exhibited a significant decrease at the 0.9 µg kg-1 treatment for both chemicals. GST (hepatopancreas) demonstrated a significant dose dependent decrease at all concentrations of both mercury chloride and dimethoate. AChE (tail muscle) decreased at the 0.6 and 0.9 µg kg-1 concentrations of dimethoate and 0.9 µg kg-1 mercury chloride. In gill tissue, MT increased in response to 0.3 and 0.6 µg kg-1 of mercury chloride but no effect was observed at the 0.9 µg kg-1 concentration of mercury chloride or any concentrations of dimethoate tested. MT did not change in response to mercury or dimethoate in tail tissue. Furthermore, neither chemical modulated GSH concentrations. Our results indicate that, apart from GSH, these markers are sensitive to the pollutants tested and that animals exposed in the wild are potentially compromised in their ability to detoxify environmental contaminants and carry out normal cellular processes.

PDGF/VEGF-Related Receptor Affects Transglutaminase Activity to Control Cell Migration During Crustacean Hematopoiesis.

The platelet-derived growth factor (PDGF) receptor, a tyrosine kinase (TK) receptor whose ligand is PDGF, is crucial in the transduction of extracellular signals into cells and mediates numerous processes, such as cell proliferation, differentiation, survival, and migration. We demonstrate the important roles of a receptor TK related to the PDGF/VEGF family protein (PVR) in controlling hematopoietic progenitor cell migration by affecting extracellular transglutaminase (TGase) activity. Pl_PVR1, GenBank accession No. KY444650, is highly expressed in hemocytes and the hematopoietic tissue (HPT). Sunitinib malate was used to block the PVF/PVR downstream pathway in HPT cell culture. The addition of Sunitinib also caused the HPT cells to increase in size and begin spreading. An increase in extracellular TGase activity on the HPT cell membrane was observed in a dose-dependent manner after treatment with Sunitinib malate. The presence of crude Ast1 provided a combinatorial beneficial effect that enhanced the number of spreading cells after inhibition of the Pl_PVR downstream signaling cascade. In addition, an increased immunoreactivity for ß-tubulin and elongation of ß-tubulin filaments were found in Pl_PVR signaling-inhibited cells. The potential roles of PVF/PVR signaling in controlling progenitor cell activity during hematopoiesis in crayfish were investigated and discussed.

A Pacifastacus leniusculus serine protease interacts with WSSV.

Serine proteases are involved in many critical physiological processes including virus spread and replication. In the present study, we identified a new clip-domain serine protease (PlcSP) in the crayfish Pacifastacus leniusculus hemocytes, which can interact with the White Spot Syndrome Virus (WSSV) envelope protein VP28. It was characterized by a classic clip domain with six strictly conserved Cys residues, and contained the conserved His-Asp-Ser (H-D-S) motif in the catalytic domain. Furthermore, signal peptide prediction revealed that it has a 16-residue secretion signal peptide. Tissue distribution showed that it was mainly located in P. leniusculus hemocytes, and its expression was increased in hemocytes upon WSSV challenge. In vitro knock down of PlcSP decreased both the expression of VP28 and the WSSV copy number in hematopoietic stem (HPT) cells. Accordingly, these data suggest that the new serine protease may be of importance for WSSV infection into hematopoietic cells.

Triosephosphate Isomerase and Filamin C Share Common Epitopes as Novel Allergens of Procambarus clarkii.

Triosephosphate isomerase (TIM) is a key enzyme in glycolysis and has been identified as an allergen in saltwater products. In this study, TIM with a molecular mass of 28 kDa was purified from the freshwater crayfish (Procambarus clarkii) muscle. A 90-kDa protein that showed IgG/IgE cross-reactivity with TIM was purified and identified as filamin C (FLN c), which is an actin-binding protein. TIM showed similar thermal and pH stability with better digestion resistance compared with FLN c. The result of the surface plasmon resonance (SPR) experiment demonstrated the infinity of anti-TIM polyclonal antibody (pAb) to both TIM and FLN c. Five linear and 3 conformational epitopes of TIM, as well as 9 linear and 10 conformational epitopes of FLN c, were mapped by phage display. Epitopes of TIM and FLN c demonstrated the sharing of certain residues; the occurrence of common epitopes in the two allergens accounts for their cross-reactivity.

Population genetic structure and molecular diversity of the red swamp crayfish in China based on mtDNA COI gene sequences.

Population genetic structure and molecular diversity are closely related to adaptability, potential and evolutionary of a species, which also reflects its population history. We analyzed the molecular variability and genetic structure among 24 populations of the red swamp crayfish in China based on the COI region. The COI of 687 bp aligned across 44 haplotypes, the average AT content (68.1%) was slightly higher than the AT content (31.9%). AMOVA indicated that a high proportion of the total genetic variance was attributable to variations within populations (87.57%), whereas only 12.43% occurred among populations. The Fst values were between 0.016 and 0.585, and the Nm values were between 0.178 and 15.182 in each population. All of the AMOVA, Fst statistics and Nm values suggested low genetic differentiation, but a high level genetic diversity existed in Chinese populations of Procambarus clarkii. The phylogenetic trees showed that some geographical populations were irregularly distributed according to the river systems while others were matched well, suggesting that genetic differentiation is created largely by geographic isolation.

Regulation of crayfish, Orconectes virilis, tail muscle lactate dehydrogenase (LDH) in response to anoxic conditions is associated with alterations in phosphorylation patterns.

Lactate dehydrogenase (LDH), the terminal enzyme of anaerobic glycolysis, has a crucial role in sustaining ATP production by glycolysis during periods of anoxia via regenerating NAD+ through the production of lactate. The present study examined the effects of prolonged (20h) anoxic submergence on LDH from the tail muscle of an anoxia-tolerant crayfish (Orconectes virilis). LDH was purified to homogeneity from tail muscle of both aerobic control and anoxic crayfish in a three step process. Analysis of the kinetic parameters and the stability of LDH showed that the Vmax in the pyruvate-reducing direction was significantly higher for the enzyme from anoxic crayfish whereas in the lactate-oxidizing direction the Vmax was significantly higher for the control enzyme. Differential scanning fluorimetry was used to assess thermal unfolding of crayfish LDH. The results showed that the enzyme from control muscle had a significantly higher melting temperature (greater thermal stability) than the anoxic enzyme form, suggesting that there was a structural difference between the two enzyme forms. Immunoblotting of purified LDH implicated post-translational modification as the reason for this difference; purified LDH from aerobic control crayfish showed significantly higher amounts of serine/threonine phosphorylation than did the anoxic enzyme form. This study provides evidence for anoxia-induced modifications of crayfish muscle LDH that may contribute significantly to modulating enzyme function under anoxic conditions.

Accumulation and detoxification dynamics of microcystin-LR and antioxidant responses in male red swamp crayfish Procambarus clarkii.

MC-LR is one of major microcystin isoforms with potent hepatotoxicity. In the present study, we aim to: 1) explore the dynamics of MC-LR accumulation and elimination in different tissues of male red swamp crayfish Procambarus clarkii; 2) reveal the mechanisms underlying hepatic antioxidation and detoxification. In the semi-static toxicity tests under the water temperature of 25±2°C, P. clarkii were exposed to 0.1, 1, 10 and 100µg/L MC-LR for 7days for accumulation and subsequently relocated to freshwater for another 7days to depurate MC-LR. MC-LR was measured in the hepatopancreas, intestine, abdominal muscle and gill by HPLC. The enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST), content of glutathione (GSH), and transcripts of Mn-sod, cat, gpx1, Mu-gst, heat shock protein90 (hsp90), hsp70 and hsp60 in hepatopancreas were detected. The results showed that P. clarkii accumulated more MC-LR in intestine, and less in abdominal muscle and gill during accumulation period and eliminated the toxin more quickly in gill and abdominal muscle, and comparatively slowly in intestine during depuration period. The fast increase of SOD and CAT activities at early stage, subsequent decrease at later stage of accumulation period and then fast increase during depuration period were partially consistent with the transcriptional changes of their respective genes. GPx was activated by longer MC-LR exposure and gpx1 mRNA expression showed uncoordinated regulation pattern compared with its enzyme. Hsp genes were up-regulated when P. clarkii was exposed to MC-LR.

Long-Term Starvation and Posterior Feeding Effects on Biochemical and Physiological Responses of Midgut Gland of Cherax quadricarinatus Juveniles (Parastacidae).

We investigated the effect of long-term starvation and posterior feeding on energetic reserves, oxidative stress, digestive enzymes, and histology of C. quadricarinatus midgut gland. The crayfish (6.27 g) were randomly assigned to one of three feeding protocols: continuous feeding throughout 80 day, continuous starvation until 80 day, and continuous starvation throughout 50 day and then feeding for the following 30 days. Juveniles from each protocol were weighed, and sacrificed at day 15, 30, 50 or 80. The lipids, glycogen, reduced glutathione (GSH), soluble protein, lipid peroxidation (TBARS), protein oxidation (PO), catalase (CAT), lipase and proteinase activities, and histology were measured on midgut gland. Starved crayfish had a lower hepatosomatic index, number of molts, specific growth rate, lipids, glycogen, and GSH levels than fed animals at all assay times. The starvation did not affect the soluble protein, TBARS, PO levels and CAT. In starved juveniles the lipase activity decreased as starvation time increased, whereas proteinase activity decreased only at day 80. The histological analysis of the starved animals showed several signs of structural alterations. After 30 days of feeding, the starved-feeding animals exhibited a striking recovery of hepatosomatic index, number of molts, lipids and glycogen, GSH, lipase activity and midgut gland structure.