Evaluation of the exploitation potential of red swamp crayfish (Procambarus clarkii) in Portugal

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente, perfil de Ordenamento do Território e Impactes Ambientais

Isolation of DNA sequences with homology to a degenerate FaRP oligonucleotide from the crayfish Procambarus clarkii

The neuropeptide Th1RFamide with the sequence Phe-Met-Arg-Phe-amide was originally isolated in the clam Macrocallista nimbosa (price and Greenberg, 1977). Since its discovery, a large family ofFl\1RFamide-related peptides termed FaRPs have been found to be present in all major animal phyla with functions ranging from modulation of neuronal activity to alteration of muscular contractions. However, little is known about the genetics encoding these peptides, especially in invertebrates. As FaRP-encoding genes have yet to be investigated in the invertebrate Malacostracean subphylum, the isolation and characterization ofFaRP-encoding DNA and mRNA was pursued in this project. The immediate aims of this thesis were: (1) to amplify mRNA sequences of Procambarus clarkii using a degenerate oligonucleotide primer deduced from the common amino acid sequence ofisolated Procambarus FaRPS, (2) to determine if these amplification products encode FaRP gene sequences, and (3) to create a selective cDNA library of sequences recognized by the degenerate oligonucleotide primer. The polymerase chain reaction - rapid amplification of cDNA ends (PCR-RACE) is a procedure in which a single gene-specific primer is used in conjunction with a generalized 3' or 5' primer to amplify copies ofthe region between a single point in the transcript and the 3' or 5' end of cDNA of interest (Frohman et aI., 1988). PCRRACE reactions were optimized with respect to primers used, buffer composition, cycle number, nature ofgenetic substrate to be amplified, annealing, extension and denaturation temperatures and times, and use of reamplification procedures. Amplification products were cloned into plasmid vectors and recombinant products were isolated, as were the recombinant plaques formed in the selective cDNA library. Labeled amplification products were hybridized to recombinant bacteriophage to determine ligated amplification product presence. When sequenced, the five isolated PCR-RACE amplification products were determined not to possess FaRP-encoding sequences. The 200bp, 450bp, and 1500bp sequences showed homology to the Caenorhabditis elegans cosmid K09A11, which encodes for cytochrome P450; transfer-RNA; transposase; and tRNA-Tyr, while the 500bp and 750bp sequences showed homology with the complete genome of the Vaccinia virus. Under the employed amplification conditions the degenerate oligonucleotide primer was observed to bind to and to amplify sequences with either 9 or 10bp of 17bp identity. The selective cDNA library was obselVed to be of extremely low titre. When library titre was increased, white. plaques were isolated. Amplification analysis of eight isolated Agt11 sequences from these plaques indicated an absence of an insertion sequence. The degenerate 17 base oligonucleotide primer synthesized from the common amino acid sequence ofisolated Procambarus FaRPs was thus determined to be non-specific in its binding under the conditions required for its use, and to be insufficient for the isolation and identification ofFaRP-encoding sequences. A more specific primer oflonger sequence, lower degeneracy, and higher melting temperature (TJ is recommended for further investigation into the FaRP-encoding genes of Procambarlls clarkii.

Response of crayfish, Procambarus clarkii, haemocytes infected by white spot syndrome virus

White spot syndrome virus ( WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultrathin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.White spot syndrome virus (WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultra-thin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.

The red swamp crayfish Procambarus clarkii in Europe: Impacts on aquatic ecosystems and human well-being

Procambarus clarkii is currently recorded from 16 European territories. On top of being a vector of crayfish plague, which is responsible for large-scale disappearance of native crayfish species, it causes severe impacts on diverse aquatic ecosystems, due to its rapid life cycle, dispersal capacities, burrowing activities and high population densities. The species has even been recently discovered in caves. This invasive crayfish is a polytrophic keystone species that can exert multiple pressures on ecosystems. Most studies deal with the decline of macrophytes and predation on several species (amphibians, molluscs, and macroinvertebrates), highlighting how this biodiversity loss leads to unbalanced food chains. At a management level, the species is considered as (a) a devastating digger of the water drainage systems in southern and central Europe, (b) an agricultural pest in Mediterranean territories, consuming, for example, young rice plants, and (c) a threat to the restoration of water bodies in north-western Europe. Indeed, among the high-risk species, P. clarkii consistently attained the highest risk rating. Its negative impacts on ecosystem services were evaluated. These may include the loss of provisioning services such as reductions in valued edible native species of regulatory and supporting services, inducing wide changes in ecological communities and increased costs to agriculture and water management. Finally, cultural services may be lost. The species fulfils the criteria of the Article 4(3) of Regulation (EU) No 1143/2014 of the European Parliament (species widely spread in Europe and impossible to eradicate in a cost-effective manner) and has been included in the “Union List”. Particularly, awareness of the ornamental trade through the internet must be reinforced within the European Community and import and trade regulations should be imposed to reduce the availability of this high-risk species.

Eradication d'une population d'écrevisses de Louisiane, Procambarus clarkii, à l'étang de Vidy et situation des populations d'écrevisses à Lausanne, Suisse.

En juillet 2007, des écrevisses de Louisiane, Procambarus clarkii, ont été observées, pour la première fois en Suisse romande, dans l'étang artificiel de Vidy à Lausanne. Au printemps 2008, l'étang a été asséché pendant 2 mois pour tenter d'éradiquer les écrevisses. En juillet 2008, et au printemps 2009, des écrevisses de Louisiane furent à nouveau observées dans l'étang: des individus avaient survécu au traitement et/ou des écrevisses avaient à nouveau été introduites. En 2010, une nouvelle campagne d'éradication a été mise en place: l'étang fut asséché, son lit chaulé et les interstices entre les enrochements bétonnés. Lors du suivi 2011, plus aucune écrevisse ne fut capturée. L'opération semble donc finalement avoir été un succès. Les observations effectuées sur 20 plans d'eau dans un rayon de 4.5 km autour de l'étang de Vidy n'ont pas mis en évidence d'autres populations d'écrevisses de Louisiane. Plusieurs populations d'écrevisses américaines, orconectes limosus, et signal ̧ Pacifastacus lenuisculus, ont été localisées, ainsi qu'une population d'écrevisses à pattes rouges, Astacus astacus.

The role of cyclic nucleotides in modulation of crayfish neuromuscular junctions by a neuropeptide /

DF2, a heptapeptide, is a member of the family of FMRFamide-like peptides and has been shown to increase the amount of transmitter released at neuromuscular junctions of the crayfish, Procambarus clarkit Recent evidence has shown that protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII) and the cAMPdependent protein kinase (PKA) play a role in the neuromodulatory pathway of DF2. The involvement of these kinases led to the prediction that a G-protein-coupled receptor (GPCR) is activated by DF2 due to the role that each kinase plays in traditional GPCR pathways seen in other organisms and in other cells. G-proteins can also act on an enzyme that generates cyclic guanosine monophosphate (cGMP) which mediates its effects through a cGMP-dependent protein kinase (PKG). This thesis addresses the question of whether or not DF2's effects on synaptic transmission in crayfish are mediated by the cyclic nucleotides cAMP and cGMP. The effects of DF2 on synaptic transmission were examined using deep abdominal extensor muscles of the crayfish Procambarus clarkii. An identified motor neuron was stimulated, and excitatory post-synaptic potentials (EPSPs) were recorded in abdominal extensor muscle LI . A number of activators and inhibitors were used to determine whether or not cAMP, PKA, cGMP and PKG mediate the effect of this peptide. Chemicals that are known to activate PKA (Sp-cAMPS) and/or PKG (8-pCPTcGMP) mimic and potentiate DF2's effect by increasing EPSP amplitude. Inhibitors of either PKA (Rp-cAMPS) or PKG (Rp-8-pCPT-cGMPS) block a portion of the increase in EPSP amplitude induced by the peptide. When both kinase inhibitors are applied simultaneously, the entire effect of DF2 on EPSPs is blocked. The PKG inhibitor blocks the effects of a PKG activator but does not alter the effect of a PKA activator on EPSP amplitude. Thus, the PKG inhibitor appears to be relatively specific for PKG. A trend in the data suggests that the PKA inhibitor blocks a portion of the response elicited by the PKG activator. Thus, the PKA inhibitor may be less specific for PKA. Phosphodiesterase inhibitors, which are known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of the peptide. These results support the hypothesis that cAMP and cGMP, acting through their respective protein kinase enzymes, mediate the ability of DFi to increase transmitter output.

Effects of reflection and social isolation on crayfish behaviour /

Visual stimuli and socialization influence exploratory behaviour in crayfish. The predominant components of spontaneous exploratory behaviour were determined by observing the activity of solitary adult crayfish (Procambarus clarkii) in a glass aquarium containing fresh water and no objects. Five distinct behaviours were observed: rearing up (climbing on the wall), turning around, cornering (facing the comer), backward walking, and crossing (crossing the midline of the aquarium). The frequency of rearing up, cornering and turning around decreased when reflection from the glass wall was blocked with black cardboard, black paint or non-reflective transparent plastic. In a tank containing mirrors on one side and non-reflective plastic on the other, crayfish cornered, reared up, and turned around more in front of the mirrors. Socialization was necessary for crayfish to respond to the reflection. Crayfish that were housed in pairs for two weeks exhibited more rearing up, turning around and cornering in front of the mirrors than in the non-reflective side. Crayfish isolated for two weeks did not show these differences. Socialized crayfish also exhibited more rearing up, turning around and cornering than did isolated crayfish. Thus, crayfish respond to visual stimuli provided by a glass tank, but the responds depends on socialization.

Tamaño poblacional, morfometría y crecimiento de Procambarus clarkii (Girard) (Crustácea: cambaridae) del área central de Nuevo León, México

Tesis (Maestría en Ciencias con Especialidad en Ecología Acuática y Pesca) U.A.N.L.

Evaluación del crecimiento y sobrevivencia del acocil rojo procambarus clarkii (GIRARD, 1852) con un alimento natura y una dieta formulada bajo condiciones de laboratorio.

Tesis (Maestría en Ciencias con Especialidad en Recursos Alimenticios y Producción Agrícola) UANL, 2012.

Fisiología reproductiva del acocil rojo Procambarus clarkii (Crustacea: decapoda) : establecimiento del ciclo de maduración gonadal y evaluación de su potencial reproductivo

Tesis (Doctorado en Ciencias Biológicas con Especialidad en Acuacultura) UANL

Morfometría, distribución actual y potencial en el norte de México del acocil rojo procambarus clarkii (Girard, 1852) (crustacea: cambaridae)

Tesis (Doctor en Ciencias con acentuación en Manejo de Vida Silvestre y Desarrollo Sustentable) UANL, 2014.

Assessment of Donana National Park contamination in Procambarus clarkii: Integration of conventional biomarkers and proteomic approaches

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Biochemical and proteomic effects in Procambarus clarkii after chlorpyrifos or carbaryl exposure under sublethal conditions

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Responses to Reflection in Two Invertebrate Species

The present thesis investigates the responses to reflection in both the crayfish Procambarus clarkii and the fruit fly Drosophila melanogaster. Responses to reflection in crayfish depend on social status and the current work suggests that learning and memory consolidation are required for these responses to be altered. Crayfish were treated to either massed or spaced training fights prior to reflection testing. The results show that subordinate crayfish treated to spaced training display a response typical of subordinate crayfish but subordinate crayfish treated to massed training exhibit a response typical of dominant crayfish. Fruit flies are shown to be attracted to reflection and responses to reflection are described here for the first time. Responses in fruit flies are shown to be dependent on social status. The frequency of behaviours were altered in isolated flies but not socialized flies. The addition of pheromones cVA and 7,11-HD were used to investigate how the addition of chemical cues altered responses to reflection in fruit flies. Socialized fruit flies treated with cVA exhibited an increase in the frequency of behaviours on both mirrored and clear glass walls, while isolated flies exhibited a decrease. Socialized flies treated with 7,11-HD spent more time on mirrored walls compared to glass walls, whereas the frequency of all behaviours were decreased in isolated flies treated with 7,11-HD.