Alexandra M. Correia

Seasonal and interspecific evaluation of predation by mammals and birds on the introduced red swamp crayfish Procambarus clarkii (Crustacea, Cambaridae) in a freshwater marsh (Portugal)

The crayfish Procambarus clarkii is an introduced species in Portugal, becoming a new available resource that has possibly changed local trophic relationships. The purpose of the present study was to understand better how a naive assembly of predators reacted to a recent invader. Predation on P. clarkii by nine species of mammals (Order: Carnivora) and six species of birds (Order: Ciconiiformes) in a marsh area in Portugal, was surveyed through the collection of faeces and pellets over a 24-month period. Seasonal variation in the consumption of this prey by different predators was evaluated, and estimation made of size and weight of P. clarkii consumed by them. Procambarus clarkii was readily preyed upon by four species of mammalian carnivores and five species of ciconiiform birds. The consumption of P. clarkii varied seasonally in both years with a trend between seasonal exploitation and its population structure and availability. Generally, the use of P. clarkii by all predators was more intense in spring, summer and autumn than in winter, and was directed towards larger and heavier individuals. These findings result from the population dynamics presented by P. clarkii in the marsh area studied here, since larger and heavier individuals were more available in spring, summer and autumn. Procambarus clarkii is an important food resource for mammals and birds and it seems to play a key role in the trophic interactions of the riparian and terrestrial communities of the marsh.

Early Changes of Neuromuscular Transmission in the SOD1(G93A) Mice Model of ALS Start Long before Motor Symptoms Onset

Amyotrophic lateral sclerosis is characterized by a progressive degeneration of the corticospinal tract motor neurons. Growing evidence suggests that degeneration may begin at the distal axon proceeding in a dying-back pattern. It seemed therefore of interest to investigate synaptic transmission at the neuromuscular junction (NMJ) in pre- and symptomatic phases of the disease. Endplate potentials (EPPs), miniatures endplate potentials (MEPPs) and giant MEPPs (GMEPPs) were recorded from innervated diaphragm muscle fibers from 4–6 and 12-15 weeks-old SOD1(G93A) mice and non-transgenic aged-matched littermates (WT). In the pre-symptomatic phase, SOD1(G93A) mice exhibited a significant increase in the mean amplitude of EPPs together with an increase in the mean quantal content of EPPs, suggesting that more acetylcholine is being released into the synaptic cleft. SOD1(G93A) mice presented a higher frequency of GMEPPs, suggestive of intracellular Ca2+ deregulation in nerve terminals. The increase in the mean amplitude of MEPPs and the decreased mean rise-time of MEPPs in SOD1(G93A) mice point to post-synaptic related changes. In the symptomatic phase, electrophysiological data showed evidence for two NMJ groups in SOD1(G93A) mice: SOD1a and SOD1b. SOD1a group presented reduced mean amplitude of both EPPs and MEPPs. The mean rise-time of MEPPs was increased, when compared to WT and to SOD1b group, indicating impairments in the neuromuscular transmission. In contrast, the neuromuscular transmission of SOD1b group was not different from age-matched WT nor pre-symptomatic SOD1(G93A) mice, being somehow in between both groups. Altogether these results show that the neuromuscular transmission of SOD1(G93A) mice is enhanced in the pre-symptomatic phase. In the symptomatic phase our results are consistent with the hypothesis that the diaphragm of SOD1(G93A) mice is undergoing cycles of denervation/re-innervation supported by mixed neuromuscular junction populations. These early changes in the neuromuscular transmission of SOD1(G93A) mice suggest that the ALS associated events start long before symptoms onset.

Niche breadth and trophic diversity: feeding behaviour of the red swamp crayfish (Procambarus clarkii) towards environmental availability of aquatic macroinvertebrates in a rice field (Portugal)

The red swamp crayfish, Procambarus clarkii (Girard 1852), an alien species in Portugal, may have dramatic effects on aquatic communities by depleting all food resources available, just after its introduction. The purpose of this study was to evaluate, through the concept of niche breadth, whether the diet of this species reflected the temporal changes of aquatic macroinvertebrate availability once it is acclimated. Petraitis’ index of niche breadth and Herrera’s trophic diversity index were used to evaluate the trophic behaviour of P. clarkii towards available resources over time and intraspecifically (size classes and sex) in a rice field in Portugal. Results from this study showed that the consumption of aquatic macroinvertebrates by P. clarkii reflected their seasonal availability. The high values of niche breadth and trophic diversity indicated resource use according to trophic availability, diversified diets and different individual exploitation of resources regardless of size or sex. These findings suggest that in habitats where P. clarkii is already acclimated, it adjusted its trophic behaviour to the seasonal availability of aquatic macroinvertebrates. The large niche breadth and high trophic diversity presented by P. clarkii enables it to successfully expand its range to new areas, when other environmental conditions are favourable, as has been observed in Portugal and worldwide.

Shifts in aquatic macroinvertebrate biodiversity associated with the presence and size of an alien crayfish

To investigate the effects of Procambarus clarkii on macroinvertebrate diversity, we conducted a mesocosm experiment simulating small pools in rice field pads after the rice season. We hypothesized that crayfish predation would negatively impact macroinvertebrate diversity, and the magnitude of this impact should vary with the size of P. clarkii. We conducted a short-term mesocosm experiment to determine macroinvertebrate diversity in the presence of three size classes and in the absence of crayfish, as well as the diet composition of crayfish from the three size classes. At the end of the experiments, the diet of crayfish was composed of the most available taxa (Culicidae, Chironomus, Tanytarsini and Orthocladinae). These results also show evidence that, in confined areas, crayfish are important predators of major rice pests such as rice Chironominae larvae. Macroinvertebrate diversity was negatively affected by crayfish presence, but the effect was inversely proportional to crayfish size. The highest diversity index was obtained in the absence of P. clarkii, and juvenile crayfish significantly reduced macroinvertebrate diversity. Thus, the impact of P. clarkii on aquatic macroinvertebrates is size dependent and may be relevant in small pools formed in rice field pads from early autumn to late winter. Overall, our findings suggest that the negative effects of P. clarkii on macroinvertebrate diversity may be particularly strong in local natural assemblages confined to puddles of water or small ponds in wetland areas.

Processes and patterns of plant destruction by crayfish: effects of crayfish size and developmental stages of rice

Interference by crayfish on the establishment of rice plants was evaluated in this study. Two experiments were performed in outdoor containers with wet-seeded rice. The purpose of the first experiment was to determine if crayfish size has a significant impact on plant destruction. Two different scales of habitat size were analysed. The second experiment was designed to determine the influence of crayfish on different developmental stages of rice and to obtain data on the type of destruction caused by crayfish. Remaining dry weight, number and dry weight of the rice seedlings (intact + damaged) and number and dry weight of the seeds (intact + damaged) were quantified in the first experiment. The second experiment quantified: (1) number of viable seeds or seedlings, (2) dry weight of the remaining rice biomass; (3) number of viable seeds; (4) number of non-viable seeds; (5) height of any damaged seedlings, (6) number of seedlings damaged, (7) height of intact seedlings, and (8) number of intact seedlings. Our findings indicate that: increasing crayfish size results in larger negative effects on rice plant establishment; and consumption is the most important cause of destruction although the effect is reduced as plants grow. Crayfish left only a very small portion of the seedlings intact at 3 and 6 days of plant development. Thus, practical implications for management are that any crayfish control mechanisms being used are expected to be particularly effective during the first 6 days of rice growth.

Predominance of Adenosine Excitatory over Inhibitory Effects on Transmission at the Neuromuscular Junction of Infant Rats

Adenosine-induced modulation of neuromuscular transmission in young (3–4-week-old) rats was evaluated. Inhibition of adenosine kinase with iodotubercidin (ITU; 10 μM), which is known to induce adenosine release, enhanced the amplitude of evoked end-plate potentials (EPPs) recorded from innervated diaphragm muscle fibers. This facilitatory effect was transformed into an inhibitory one upon blockade of adenosine A2A receptors with 4-(2-[7-amino-2-(2-furly)[1,2,4]triazolo[2,3-a][1,3,5]triazin5ylamino] ethyl) phenol (ZM 241385) (50 nM); further blockade of adenosine A1 receptors with the selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM) abolished that inhibition. Adenosine or 2-chloroadenosine (CADO), at submicromolar concentrations, increased the amplitude and the quantal content of EPPs, whereas at low micromolar concentrations they decreased EPP amplitude. Blockade of A1 receptors with DPCPX (10 nM) prevented both excitatory and inhibitory effects, whereas blockade of A2A receptors with ZM241385 (50 nM) prevented only the excitatory effects. DPCPX and ZM241385 also prevented the excitatory effect of the selective A2A receptor agonist 2-[p-(2-carboxyethyl) phenethylamino]-5′-N-ethylcarboxamido adenosine hydrochloride (CGS 21680; 10 nM). CADO (30 nM) also increased neuromuscular transmission in adult (12–16-week-old) rats. It is suggested that at the motor nerve endings, low extracellular concentrations of adenosine activate both A2A and A1 receptors, but activation of A2A receptors predominates over A1 receptors; the activity of A2A receptors might, however, require coactivation of A1 receptors. This facilitatory action of low concentrations of extracellular adenosine upon acetylcholine release may be particularly relevant at developing neuromuscular junctions, where subtle changes in synaptic levels of acetylcholine might influence synaptic stabilization.

Adenosine A2A receptors activation facilitates neuromuscular transmission in the pre-symptomatic phase of the SOD1(G93A) ALS mice, but not in the symptomatic phase.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease leading to motor neuron dysfunction resulting in impairment of neuromuscular transmission. A2A adenosine receptors have already been considered as a potential therapeutical target for ALS but their neuromodulatory role at the neuromuscular junction in ALS remains to be clarified. In the present work, we evaluated the effects of A2A receptors on neuromuscular transmission of an animal model of ALS: SOD1(G93A) mice either in the pre-symptomatic (4–6 weeks old) or in the symptomatic (12–14 weeks old) stage. Electrophysiological experiments were performed obtaining intracellular recordings in Mg2+ paralyzed phrenic nerve-hemidiaphragm preparations. Endplate potentials (EPPs), quantal content (q. c.) of EPPs, miniature endplate potentials (MEPPs) and giant miniature endplate potential (GMEPPs) were recorded. In the pre-symptomatic phase of the disease (4–6 weeks old mice), the selective A2A receptor agonist, CGS 21680, significantly enhanced (p<0.05 Unpaired t-test) the mean amplitude and q.c. of EPPs, and the frequency of MEPPs and GMEPPs at SOD1(G93A) neuromuscular junctions, the effect being of higher magnitude (p<0.05, Unpaired t-test) than age-matched control littermates. On the contrary, in symptomatic mice (12–14 weeks old), CGS 21680 was devoid of effect on both the amplitude and q.c. of EPPs and the frequency of MEPPs and GMEPPs (p<0.05 Paired t-test). The results herein reported clearly document that at the neuromuscular junction of SOD1(G93A) mice there is an exacerbation of A2A receptor-mediated excitatory effects at the pre-symptomatic phase, whereas in the symptomatic phase A2A receptor activation is absent. The results thus suggest that A2A receptors function changes with ALS progression.

Neuromuscular transmission modulation by adenosine upon aging

In infant rats adenosine A(2A) receptor-mediated modulation of neuromuscular transmission predominates over A1 receptor-mediated neuromodulation. We investigated whether aging affects this A(2A)/A(1) receptor balance. Evoked (EPPs) and miniature end plate potentials (MEPPs) were recorded from single fibers of (weeks-old) infant (3-4), young adult (12-16), older (36-38), and aged (80-90) male rat-diaphragm. The non A1/A(2A) selective agonist, 2-chloroadenosine (CADO; 30 nM) and the adenosine kinase inhibitor, iodotubericidin (ITU; 10 μM) increased mean amplitude and quantal content of EPPs in infant, young adult, and older adult rats, but not in aged rats. The facilitatory effects were prevented by the A(2A) receptor antagonist, ZM241385 (50 nM) and mimicked by the A(2A) receptor agonist, CGS21680 (10 nM). The A1 receptor agonist, 6-cyclopentyladenosine (CPA; 100 nM), decreased EPPs amplitude in all age groups. It is concluded that aging differently influences adenosine A1 receptor and A(2A) receptor-mediated presynaptic modulation of neuromuscular transmission, so that the facilitatory influence decreases upon aging, whereas the inhibitory influence remains unchanged in aged animals. The reduction of adenosine A(2A) receptors upon aging may contribute to the age-related changes in neuromuscular function.

Seasonal and Circadian Foraging Activity of Procambarus Clarkii (Decapoda, Cambaridae) in Portugal

The feeding chronology of Procambarus clarkii has never been described. Consequently, the author (1) presents data on seasonal and circadian foraging activity of this species, (2) tests statistically the observed patterns, and (3) attempts to determine if the foraging periodicity registered here coincides with other rhythms reported elsewhere for this species. Results suggest the existence of seasonal and bimodal circadian foraging patterns. Seasonally, feeding intensity increased over the spring and summer and declined during autumn and winter. Diurnal patterns revealed increased foraging at night and a decrease in the day time. The differences were not significant. Foraging periodicity of P clarkii is coincident with locomotor rhythms and it seems to result from the interaction between specific endogenous and exogenous factors.

Influence of chemical and visual stimuli in food-search behaviour of Procambarus clarkii under clear conditions

Chemical communication may play a major role in aquatic environments because of visibility limitations, but when turbidity is reduced do chemical and visual stimuli interact to elicit food-search behaviour by Procambarus clarkii? Does P. clarkii use visual cues to detect its prey? We conducted behavioural experiments in a Y maze, under non-turbid conditions. In the experiments P. clarkii was exposed to chemical plus visual (CV) and visual only (V) stimuli transmitted by Chironomus riparius. The results revealed that P. clarkii detected CV and V cues associated with the presence of C. riparius. In controls, with no prey, crayfish displayed only searching behaviour, whereas in treatments they exhibited searching, detection and attack behaviours. Further, there were no significant differences between CV and V stimuli in the mean number of attacks performed by crayfish. Thus, our findings indicate that under low turbidity, visual cues are sufficient to mediate prey detection by P. clarkii.