Analysis of the immune transcriptome of the invasive pest spotted wing drosophila infected by Steinernema carpocapsae.

Drosophila suzukii is a pest of global concern due to its great impact on several crops. The entomopathogenic nematode Steinernema carpocapsae was highly virulent to the larvae of the fly although some immune mechanisms were triggered along the infection course. Thus, to understand the gene activation profile we performed a comparative transcriptome of D. suzukii larvae infected with S. carpocapsae and Xenorhabdus nematophila to map the differentially expressed genes involved in the defence response. The analysis exposed the induction of genes involved in the humoral response such as the antimicrobial peptides and pattern-recognition receptors while there was a suppression of the cellular defence. Besides, genes involved in melanisation, and clot formation were downregulated hindering the encapsulation response and wound healing. After the infection, larvae were in a stress condition with an enrichment of metabolic and transport functionalities. Concerning the stress response, we observed variations of the heat-shock proteins, detoxification, and peroxidase enzymes. These findings set a genetical comprehensive knowledge of the host-pathogen relation of D. suzukii challenged with S. carpocapsae which could support further comparative studies with entomopathogenic nematodes.

Biocontrol potential of six Heterorhabditis bacteriophora strains isolated in the Azores Archipelago.

Entomopathogenic nematodes (EPNs) are closely associated with Popillia japonica and potentially used as their biological control agents, although field results proved inconsistent and evoked a continual pursuit of native EPNs more adapted to the environment. Therefore, we surveyed the Azorean Archipelago to isolate new strains of Heterorhabditis bacteriophora and to evaluate their virulence against the model organism Galleria mellonella under laboratory conditions. Six strains were obtained from pasture and coastal environments and both nematode and symbiont bacteria were molecularly identified. The bioassays revealed that Az172, Az186, and Az171 presented high virulence across the determination of a lethal dose (LD50) and short exposure time experiments with a comparable performance to Az29. After 72 hours, these virulent strains presented a mean determination of a lethal dose of 11 infective juveniles cm-2, a lethal time (LT50) of 34 hours, and achieved 40% mortality after an initial exposure time of only 60 minutes. Az170 exhibited an intermediate performance, whereas Az179 and Az180 were classified as low virulent strains. However, both strains presented the highest reproductive potential with means of 1700 infective juveniles/mg of larvae. The bioassays of the native EPNs obtained revealed that these strains hold the potential to be used in biological control initiatives targeting P. japonica because of their high virulence and locally adapted to environmental conditions.

The modulation effect of the Steinernema carpocapsae - Xenorhabdus nematophila complex on immune-related genes in Drosophila suzukii larvae.

Larvae of the invasive pest Drosophila suzukii are susceptible to the Steinernema carpocapsae - Xenorhabdus nematophila complex and an assessment of the immune-regulatory system activation in this insect was performed to understand the response to the nematode infection. The expressions of 14 immune-related genes of different pathways (Imd, Toll, Jak-STAT, ProPO, JNK, TGF-ß) were analyzed using qRT-PCR to determine variations after nematode penetration (90 min and 4 h) and after bacterial release (14 h). Before the bacteria were present, the nematodes were not recognized by the immune system of the larvae and practically none of the analyzed pathways presented variations when compared with the non-infected larvae. However, after the X. nematophila were released, PGRP-LC was activated leading to the gene upregulation of antimicrobial peptides of both the Toll and Imd pathways. Interestingly, the cellular response was inactive during the infection course as Jak/STAT and pro-phenoloxidase genes remained unresponsive to the presence of both pathogens. These results illustrate how D. suzukii immune pathways responded differently to the nematode and bacteria along the infection course.

How does the beach ecosystem change without tourists during COVID-19 lockdown?

Urban tourist beach ecosystems provide the essential service of recreation. These ecosystems also support critical ecological functions where biodiversity conservation is not usually a priority. The sudden lockdown due to the COVID-19 pandemic created a unique opportunity to evaluate the effects of human absence in these urban-coastal ecosystems. This study examined bioindicators from 29 urban tourist beaches in seven Latin-American countries and assesses their response to lockdown about some relevant anthropogenic stressors such as pollution, noise, human activities, and user density. The presence of animals and plants, as well as the intensity of stressors, were assessed through a standardized protocol during lockdown conditions. Additionally, the environmental conditions of the beaches before and during lockdown were qualitatively compared using multivariate non-parametric statistics. We found notable positive changes in biological components and a clear decrease in human stressors on almost all the beaches. Dune vegetation increased on most sites. Similarly, high burrow densities of ghost crabs were observed on beaches, except those where cleaning activity persisted. Because of the lockdown, there was an exceptionally low frequency of beach users, which in turn reduced litter, noise and unnatural odors. The observed patterns suggest that tourist beaches can be restored to natural settings relatively quickly. We propose several indicators to measure changes in beaches once lockdown is relaxed. Adequate conservation strategies will render the recreational service of tourist beaches more environmental-friendly.

Effect of a gradually increasing temperature on the behavioural and physiological response of juvenile Hippocampus erectus: Thermal preference, tolerance, energy balance and growth.

The physiological and behavioural responses of ectotherms to temperature is strongly dependent on the individuals' previous thermal history. Laboratory based studies investigating the mechanisms of thermoregulation in marine ectotherms, however, rarely consider key temporal elements of thermal exposure, such as the rate at which temperature changes. We tested the hypothesis that juvenile seahorses, Hippocampus erectus, from a tropical coastal lagoon in Yucatan, Mexico, would exhibit variations in physiological and behavioural descriptors of thermoregulation when submitted to contrasting regimes during 30 days: temperature constant at 25 °C (C 25); gradually increasing 1 °C every 5 days from 25 to 30 °C (GI 25-30); and constant at 30 °C (C 30). Immediately after exposure, critical maximum temperature, thermal preference, oxygen consumption, partial energy balance, growth rate and survival of seahorses were measured. Seahorses exposed to GI 25-30 showed a significantly higher critical thermal maxima (37.8 ±â€¯0.9 °C), preference (28.7 ±â€¯0.4 °C), growth (1.10 ±â€¯0.49%) and survival (97.6%) than those exposed to C 30 (36.5 ±â€¯1, 29.4 ±â€¯0.3 °C, 0.48 ±â€¯0.32%, 73.8%, respectively). Both high temperature regimes induced metabolic depression, but ramping resulted in a greater amount of energy assimilated (278.9 ±â€¯175.4 J g-1 day-1) and higher energy efficiency for growth (89.8%) than constant exposure to 30 °C (115.4 ±â€¯63.4 J g-1 day-1, 65.3%, respectively). Gradually increasing temperature allowed physiological mechanisms of thermal adjustment to take place, reflecting the capacity of juvenile H. erectus to respond to environmental change. Despite its advantage, this capacity is limited in time, since the cumulative effect of thermal exposure affected metabolic performance, eventually compromising survival. The study of seahorse response to thermal variations in the context of ocean warming needs to consider the temporal elements of thermal exposure to foresee its vulnerability under future scenarios.

Biochemical study and in vitro insect immune suppression by a trypsin-like secreted protease from the nematode Steinernema carpocapsae.

A trypsin-like serine protease was purified by gel filtration and anion-exchange chromatography from the excretory-secretory products of parasitic phase Steinernema carpocapsae. The purified protease exhibited a molecular mass of about 29 kDa by SDS-PAGE and displayed a pI of 6.3. This protease exhibited high activity with trypsin-specific substrate N-Ben-Phe-Val-Arg-p-nitroanilide and was highly sensitive to aprotinin and benzamidine. The purified trypsin protease digested the chromogenic substrate N-Ben-Phe-Val-Arg-p-nitroanilide with K(m), V(max) and k(cat) values of 594.2 mum, 0.496 mum/min and 22.8/s, respectively. The optimal pH and temperature for protease activity were 9 and 30 degrees C, respectively. Internal amino acid sequencing yielded 150 amino acids and these were homologous to other trypsin sequences. In vitro investigation was carried out to monitor prophenoloxidase suppression in Galleria mellonella by the purified protease; about 38.9-52.6% suppression of prophenoloxidase was observed. The purified protease affected insect haemocyte spreading, causing cells to become spherical or round. Protease-treated actin filaments were highly disorganized in haemocytes. In vitro, G. mellonella haemocytes recognized infective juveniles of Heterorhabditis bacteriophora; however, S. carpocapsae and Steinernema glaseri were not recognized. We provide experimental evidence that the purified trypsin has the potential to alter host haemocytes, actin filaments and to inhibit host haemolymph melanization.

Sensitivity analysis of surface runoff generation in urban flood forecasting.

Reliable flood forecasting requires hydraulic models capable to estimate pluvial flooding fast enough in order to enable successful operational responses. Increased computational speed can be achieved by using a 1D/1D model, since 2D models are too computationally demanding. Further changes can be made by simplifying 1D network models, removing and by changing some secondary elements. The Urban Water Research Group (UWRG) of Imperial College London developed a tool that automatically analyses, quantifies and generates 1D overland flow network. The overland flow network features (ponds and flow pathways) generated by this methodology are dependent on the number of sewer network manholes and sewer inlets, as some of the overland flow pathways start at manholes (or sewer inlets) locations. Thus, if a simplified version of the sewer network has less manholes (or sewer inlets) than the original one, the overland flow network will be consequently different. This paper compares different overland flow networks generated with different levels of sewer network skeletonisation. Sensitivity analysis is carried out in one catchment area in Coimbra, Portugal, in order to evaluate overland flow network characteristics.

Real-time forecasting urban drainage models: full or simplified networks?

Lead time between rainfall prediction results and flood prediction results obtained by hydraulic simulations is one of the crucial factors in the implementation of real-time flood forecasting systems. Therefore, hydraulic simulation times must be as short as possible, with sufficient spatial and temporal flood distribution modelling accuracy. One of the ways to reduce the time required to run hydraulic model simulations is increasing computational speed by simplifying the model networks. This simplification can be conducted by removing and changing some secondary elements using network simplification techniques. The emphasis of this paper is to assess how the level of urban drainage network simplification influences the computational time and overall simulation results' accuracy. The models used in this paper comprise a sewer network and an overland flow drainage system in both 1D/1D and 1D/2D approaches. The 1D/1D model is used as the reference model to generate several models with different levels of simplifications. The results presented in this paper suggest that the 1D/2D models are not yet suitable to be used in real-time flood prediction applications due to long simulation time, while on the other hand, the simplified 1D/1D models show that considerable reductions in simulation time can be achieved without compromising simulation results (flow and water depth) accuracy.

Distribution patterns, carbon sources and niche partitioning in cave shrimps (Atyidae: Typhlatya).

Cave shrimps of the Typhlatya genus are common and widespread in fresh, brackish and marine groundwater throughout the Yucatan Peninsula (Mexico). These species are ideal models to test niche partitioning within sympatric species in oligotrophic systems. Nevertheless, their food sources remain unidentified, and despite their frequency and functional importance, distribution and abundance patterns of these species within caves have not been fully recognized. Here, we describe the abundance of three Typhlatya species in different temporal and spatial scales, investigate changes in water conditions, and potential sources of carbon as an indication of food origin. Species composition and abundance varied markedly in space and time revealing patterns that differed from one system to another and in relation to environmental parameters. Isotope analysis showed that each species reflects a particular δ13C and Δ14C fingerprint, suggesting they feed in different proportions from the available carbon sources. Overall, our findings suggest a niche partitioning of habitat and feeding sources amongst the three Typhlatya species investigated, where environmental characteristics and physiological differences could play an important role governing their distribution patterns.

Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Bovine oocyte quality in relation to ultrastructural characteristics of zona pellucida, polyspermic penetration and developmental competence.

In the present study, the effect of bovine oocyte quality related to ultrastructural characteristics of zona pellucida (ZP), polyspermic penetration and embryo developmental competence was evaluated. Cumulus-oocyte complexes were punctioned from 453 ovaries, classified as 1, 2, 3 and 4 according to their morphological aspect, matured for 24 h and then divided into two groups. In group A, oocytes were fixed in 2.5% glutaraldehyde and 0.1 m sodium cacodylate and examined under a scanning electron microscope. Photomicrographs were taken and ZP's pores were evaluated in squares of 6.4-microm width. In group B, oocytes were fertilized in vitro. After 48 h, non-cleaved oocytes were fixed for polyspermy evaluation. On days 7, 9 and 10, embryos were classified as developed (blastocysts and hatched blastocysts). Results showed that quality 1 oocytes revealed a ZP pore diameter of 0.50 +/- 0.07 microm, which was smaller than the observed on oocytes of quality 2 (0.83 +/- 0.10 microm), quality 3 (1.02 +/- 0.22 microm) and quality 4 (1.38 +/- 0.59 microm) (p < or = 0.05). For In Vitro Fertilization (IVF), results showed that embryos originating from oocytes classed as 3 and 4 had lower cleavage rate (68.4% and 43.8%) than those belonging to class 1 and 2 (79.5% and 69.3%) (p < or = 0.05). None oocyte classified as 3 and 4 developed to hatch blastocysts, while for oocytes belonging to quality 1 and 2, these values were, respectively, 15.2% and 12.5%. Concerning polyspermy, oocytes class 1 and 2 had lower polyspermic penetration than those belonging to class 3 and 4 (respectively 4.1%, 4.5%, 11.1% and 9.8%, for class 1, 2, 3 and 4). In conclusion, the present study demonstrated that oocytes with low qualities result in lower developmental competence and with high percentage of polyspermy after IVF, which can be the result of the ZP structure such as the number and the pore's diameter.

Identification of chickpea seed proteins resistant to simulated in vitro human digestion.

Proteins and peptides able to resist gastrointestinal digestion and reach the intestinal mucosa have the potential to influence human health. Chickpea (Cicer arietinum L.) seed proteins are able to resist cooking (86.9% total protein) and/or in vitro simulated human digestion (15.9% total protein resists soaking, cooking and digestion with pepsin and pancreatin). To identify and characterize proteins resisting digestion we made use of different MS methodologies. The efficiency of several proteases (trypsin, AspN, chymotrypsin and LysC) was tested, and two technologies were employed (MALDI-MS/MS and LC-nESI-MS/MS). Digestion with trypsin and AspN were most successful for the identification of seed proteins. When analyzed by MALDI- MS/MS, trypsin allowed the identification of at least one protein in 60% of the polypeptide bands, while AspN allows the identification in 48%. The use of LC-nESI-MS/MS, allowed the identification of much more proteins/polypeptides from digested seeds (232 vs 17 using trypsin). The majority of the proteins found to be able to resist simulated digestion were members of the 7S vicilin and 11S legumin seed storage protein classes, which are reported to contain bio-active functions. In addition, we have found proteins that had not yet been described as potentially able to cause an impact on human health. SIGNIFICANCE: This is the first proteomic study to analyze the effect of processing and simulated human gastrointestinal digestion on the proteome of chickpea seed. Chickpea is reported to have anti-nutritional effects as well as nutraceutical properties, so the identification and characterization of the proteins able to resist digestion is crucial to understand the targets underlying such properties.

Purification and characterization of two serine protease inhibitors from the hemolymph of Mythimna unipuncta.

Two serine protease inhibitors, trypsin inhibitor and alpha-chymotrypsin inhibitor, were isolated from the hemolymph of Mythimna unipuncta. Mythimna trypsin and alpha-chymotrypsin inhibitors were purified by gel filtration and anion-exchange chromatography. They displayed molecular masses of 52 kDa and 43 kDa, respectively, as determined by electrophoresis under reducing and non-reducing conditions on denaturing polyacrylamide gels. Their isoelectric points were evaluated by isoelectric focusing and two-dimensional electrophoresis. Their N-terminal sequences have been analyzed as APSDTTIAETLTITEEFFPD and FDESFGFQGPSTYEKTPLGEP, respectively. The role of these inhibitors in the regulation of the defense reaction of the insect is discussed.

Assessment of fluorescent-labeled bacteria for evaluation of in vivo uptake of bacteria (Vibrio spp.) by crustacean larvae.

Available methods to study crustacean digestive tract colonization by bacteria are laborious, time-consuming, and do not permit in vivo assays and observation. This paper reports on a rapid and consistent technique to apply a fluorescent label to bacteria, which can then be presented to filter-feeding crustacea such as Artemia and penaeid larvae for later in situ bacterial distribution observation. Three luminescent Vibrio spp. were stained and observed inside Artemia nauplii, shrimp zoea and mysis stages, Vibrio harveyi type strain ATCC 14126, M(1) (pathogenic) and Ea (non-pathogenic). Factors such as dye (DTAF) concentration, exposure time/temperature and sonication time were evaluated. Viability of the dye and stained bacteria were tested at 4, -20 and -70 degrees C storage temperatures for up to 81 days. Results show that 4 and -20 degrees C storage temperatures are not recommended. At -70 degrees C, both bacteria and dye are optimally preserved. Monodispersed fluorescent-labeled bacterial cells can be observed inside the digestive tract of crustacean larvae at a density of inoculation as high as 5.2 x 10(6) CFU ml(-1). After 2 to 4 h, some leaching occurs, increasing difficulty in observation, although after 24 h, it is still possible to observe monodispersed FLB inside the digestive tract of crustacean larvae. Autofluorescence may complicate observation when filter-feeding crustacean larvae are co-fed with microalgae.

Susceptibility of Pseudaletia unipuncta (Lepidoptera: Noctuidae) to entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) isolated in the Azores: effect of nematode strain and host age.

The armyworm, Pseudaletia unipuncta (Haworth), is a serious pest to the Azores's pastures. In laboratory bioassays we tested the susceptibility of this insect to entomopathogenic nematodes isolated in Azores: Steinernema carpocapsae Az20, Az150, and A48 strains, S. glaseri Az26 strain and Heterorhabditis bacteriophora Az33 strain. The A48, Az20, and Az150 strains caused parasitism rates of 96.6, 90, and 53.3%, and mortality rates of 63.3, 46.6, and 23.3%, respectively, to sixth instar. The Az33 strain caused a parasitism rate of 73.3% and a mortality rate of 40%; whereas, the Az26 strain caused a parasitism rate of 40% and no mortality. A linear response dose-parasitism with a positive regression (r2 = 0.993) was observed in insects exposed to S. carpocapsae Az150 strain. Positive regressions were also observed between mortality and dose rate for S. carpocapsae A48 (r2 = 0.980), Az20 (r2 = 0.956), and Az150 (r2 = 0.963) strains, and H. bacteriophora Az33 strain (r2 = 0.999). Fourth instars were the most susceptible to the A48 strain, followed by the fifth instars, while the sixth instars were the less susceptible, with LD50 values of 26.2, 62.8, and 320.7 infective juveniles, respectively. The lethal time for each of the tested instars was 32.3, 35.5, and 49.2 h, respectively. The invasion rate was 33.5, 28.2, and 40.8 nematodes per treated larvae in the fourth, fifth, and sixth instars, respectively.

Effectiveness of Steinernema spp. and Heterorhabditis bacteriophora against Popillia japonica in the Azores.

Steinernema carpocapsae (Breton strain), S. glaseri, and Heterorhabditis bacteriophora were evaluated for their potential to control immature stages of the Japanese beetle, Popillia japonica, on Terceira Island (the Azores). In bioassays carried out at temperatures higher than 15 C, S. glaseri and H. bacteriophora caused 100% mortality of larvae, whereas S. carpocapsae caused 56% larval mortality. At temperatures slightly below 15 C, only S. glaseri remained effective. In field plots, in September, S. glaseri and S. carpocapsae reduced larval populations by 91% and 44%, respectively, when applied at the rate of 10 nematodes/m(2). In April, S. glaseri caused 31% reduction in numbers of larvae, but S. carpocapsae was ineffective. In colder months (November-February) neither steinernematids nor H. bacteriophora reduced larval populations. Increasing the application rate from 10 to 5 x 10 infective stage S. glaseri per m(2) increased efficacy from 63% to 79% mortality.

Natural occurrence of entomopathogenic nematodes (rhabditida: steinernema, heterorhabditis) in the azores.

A soil survey for entomopathogenic nematodes was conducted throughout the nine islands of the Azorean archipelago. Forty-six out of 1,180 samples (3.9%) were positive, with Heterorhabditis spp. isolated from 30 sites on six islands and Steinernema spp. isolated from 16 sites on three islands. São Miguel and Terceira Islands were positive for both genera, and Pico Island was positive only for Steinernema. Entomopathogenic nematodes were found from sea level up to 750 m. Seventy percent of the samples positive for Heterorhabditis were collected below 150 m, whereas 62.5% of the samples positive for Steinernema were collected above 300 m. Heterorhabditis was not isolated above 450 m. Steinernema was collected mostly in loamy-sand and sandy-loam soils with a pH below 6, whereas Heterorhabditis was mostly collected in sandy and loamy-sand soils with pH higher than 6. Steinernema and Heterorhabditis were found in cropland, orchards, and pastures, while Heterorhabditis was found also in woodland and native vegetation.

Rancho flotation bed.

The Rancho Flotation Bed provides hydrostatic support with maximum pressures over bony prominences of 15 to 25 mm Hg (measured with a pneumatic pressure transducer). This is generally below the levels normally quoted as conducive to the development of ischaemia. Clinical experience has shown the bed to be a successful aid to nursing by eliminating the need to turn the patients for pressure reasons, allowing patients with pressure sores to remain in a position which is more comfortable and more suitable for other nursing care. It also makes it easier for nurses to handle patients in order to care for the pressure sores.

Structure of the testate amoebae community in different habitats in a neotropical floodplain.

This study evaluated the differences in composition, abundance and morphology of testate amoebae among different habitats of the same aquatic environment (plankton, aquatic macrophyte and sediment) in the Upper Paraná River floodplain. Triplicate samplings were undertaken monthly at each habitat from April 2007 to March 2008. The structure of the community of testate amoebae was different among the habitats. The species typical for each habitat, according to Indval, were classified by their shell morphology. Arcella species together with Difflugia gramen and Difflugia pseudogramem were more abundant for plankton. Trinema and Phryganella stood out by their abundance and frequency in aquatic macrophytes. Centropyxis was an indicator of sediment. The results indicated a higher frequency of hemispherical and spherical shells in plankton and spherical and elongated shells in aquatic macrophytes. In the sediment, there was a high frequency of elongated species. Our results support the hypothesis that the community of testate amoebae has different structures among the habitats, refuting the idea that the organization of this community in plankton is guided by random events like the resuspension of organisms from the sediment and their displacement from marginal vegetation.

Effects of recurrent disturbances on the periphyton community downstream of a dammed watercourse.

We evaluate experimentally the effect of a sequential disturbance-desiccation-on the structure and dynamics of a periphytic algal community in a semilotic environment of the Upper Paraná River floodplain. We tested the hypothesis that the presence of recurrent disturbances have a direct negative effect on the attributes of the periphyton community. The sequential effect of desiccation on the periphytic community promoted its significant decrease in density, while the same was not observed in species richness. When desiccation was induced in a mature community, there was no difference in the community compared to control. The sequential disturbances on the community of periphytic algae in a mature stage, was characterised by greater stability. It is believed that the effects of variation in water levels caused by upstream reservoirs can likewise also modify the structure and stability of periphytic algae in the Upper Paraná River floodplain.