Subtype Distribution of Blastocystis spp. in Patients with Gastrointestinal Symptoms in Northern Spain.

Limited molecular data exist on the prevalence and subtype distribution of Blastocystis spp., the most prevalent parasite in human and animal feces worldwide. A total of 44 different subtypes (STs) of Blastocystis are currently recognized based on the sequence of the small subunit ribosomal RNA (SSU-rRNA) gene. This is a molecular study of Blastocystis spp. in hospitalized patients with gastrointestinal symptoms in northern Spain. We analyzed 173 Blastocystis-positive patients with gastrointestinal symptoms by using nested PCR for molecular detection, subtype identification, phylogenetic analyses, and genetic diversity assessment. ST2 (34.1%) and ST3 (34.7%) predominated, followed by ST1 (15.6%) and ST4 (15.6%). Mixed infections with different subtypes were observed in some patients. Sequence analysis revealed for the first time in European humans the allele 88 (a variant of ST1). In other cases, alleles commonly found in animal samples were detected (allele 9 in ST2, allele 34 in ST3, and allele 42 in ST4). Phylogenetic analysis showed high variability in ST1 and ST2, suggesting a polyphyletic origin, while both ST3 and ST4 exhibited higher genetic homogeneity, indicating a possible monophyletic origin and recent transmission to humans. These data confirm Blastocystis spp. subtype diversity and may help in understanding the evolutionary processes and potential zoonotic transmission of this parasite.

Influence of mechanical and manual pruning on the incidence of pests in 'Clemenules' mandarins.

BACKGROUND: In Spain, citrus pruning is usually done by hand, although mechanized pruning is gradually being introduced as a cheaper alternative. The pruning strategy affects the pattern and intensity of sprouting, and the canopy characteristics, and therefore could affect pest control. In this study, the effect of three pruning strategies on the incidence of key citrus pests was analyzed: manual, mechanical (with hedging and topping) and no pruning (control). In a commercial clementine orchard, sprouting, pest density and fruit damage were evaluated through three seasons. RESULTS: Outside the canopy, mechanically pruned trees had a significantly higher number of shoots, and more shoots were attacked by aphids (cotton aphid, Aphis gossypii, and spirea aphid, A. spiraecola) in comparison with manual or control strategies. Inside the canopy statistically nonsignificant differences were found between strategies. Regarding the pest level of two-spotted spider mite, Tetranychus urticae, and California red scale, Aonidiella aurantii, in general, no significant differences were found between pruning strategies, and in some cases mechanical pruning resulted in a lower level of these pests and fruit damage than manual pruning. CONCLUSION: The density of aphids, which are pests associated with sprouting, was affected by the pruning strategy. However, the densities of T. urticae and A. aurantii and the level of damaged fruit were unaffected. © 2023 Society of Chemical Industry.

Contribution to optimization and standardization of antibacterial assays with silver nanoparticles: the culture medium and their aggregation.

The antimicrobial activity of silver nanoparticles is determined by their size and specific properties, as well as by the chemical composition of the exposure medium in which the nanoparticles are suspended. When the antibacterial tests are carried out in a culture medium, aggregation of the nanoparticles is produced, decreasing their effectiveness. This study proposes the addition of surfactants to the culture medium to prevent the aggregation of silver nanoparticles and optimizes the concentrations of these surfactants. The aggregation of silver nanoparticles was studied by dynamic light scattering (DLS) after dispersion in three liquid culture media (Mueller-Hinton (MH), Luria-Bertani (LB) and Brain Heart Infusion) in which four different surfactants (SDS, Triton X100, Tween 80 and CTAB) were added at concentrations of 0, 0.1, 0.5, 1, 1.5 and 2%. Results showed that, the optimal culture media to prevent aggregation of silver nanoparticles were MH and LB with higher concentrations of Tween 80 and Triton X100 surfactants; being MH + 2% of Tween 80 and MH + 1% Triton X100 the best combinations obtained because the results obtained were closest to the sizes of nanoparticles in ultrapure water. In addition, it has been verified that the optimal medium + surfactant combinations chosen did not affect the viability of Escherichia coli bacteria. Nanoparticle aggregation was not observed by single particle inductively coupled plasma mass spectrometry (SP-ICP-MS) when nanoparticles were incubated for long incubations periods (24 h) in the optimal medium chosen.

Identification of free-living amoebas and amoeba-resistant bacteria accumulated in Dreissena polymorpha.

To identify the free-living amoeba (FLA) and amoeba-resistant bacteria (ARB) accumulated in zebra mussels and in the water in which they are found, mussels were collected at two locations in the Ebro river basin (North East Spain). FLAs and bacteria were isolated from mussel extracts and from natural water. PCR techniques were used to identify the FLAs and endosymbiont bacteria (Legionella, Mycobacterium, Pseudomonas and cyanobacteria), and to detect Giardia and Cryptosporidium. The most frequently found FLAs were Naegleria spp. The presence of Legionella, Mycobacterium and Pseudomonas inside the FLA was demonstrated, and in some cases both Legionella and Pseudomonas were found together. Differences between FLAs and ARB identified inside the mussels and in the water were detected. In addition, Escherichia coli, Clostridium perfringens, Salmonella spp. and Enterococcus spp. were accumulated in mussels in concentrations unconnected with those found in water. The results show the ability of the zebra mussel to act as a reservoir of potentially pathogenic FLAs, which are associated with potentially pathogenic ARB, although the lack of association between microorganisms inside the mussels and in the water suggests that they are not useful for monitoring microbiological contamination at a specific time.

Seeking the reuse of effluents and sludge from conventional wastewater treatment plants: Analysis of the presence of intestinal protozoa and nematode eggs.

Some of the microorganisms present in urban wastewater, which include intestinal protozoa and nematodes, can be pathogenic. Their (oo)cyst and egg transmissible stages are very resistant to environmental stresses and disinfectants and they are therefore difficult to remove. Thus, they can constitute a health risk if water or sludge obtained in the purification of wastewater is reused for agricultural purposes. In this context, the presence of intestinal protozoa and nematodes were studied in influents, effluents and sludge from five wastewater treatment plants (WWTPs) in the north of Spain by optical microscopy and PCR techniques. The removal efficiency of different wastewater treatments was also compared. The presence of protozoa has increased among the population discharging waste to WWTPs in recent years. Cryptosporidium spp., Giardia duodenalis, Entamoeba spp. and nematodes were detected in all of the WWTPs. Indeed, this is the first report of Entamoeba histolytica and Entamoeba moshkovskii in Spanish WWTPs. The water treatments studied showed different removal efficiencies for each species of intestinal protozoa, with the aerated lagoons providing the best results. (Oo)cysts were also detected in sludge even after aerobic digestion and dehydration. To avoid risks, (oo)cyst viability should be analysed whenever the sludge is to be used as a fertilizer. This study reinforces the necessity of establishing legal limits on the presence of protozoa in WWTP effluents and sludges, especially if reuse is planned. Further studies are necessary for a better understanding of the presence and behaviour of intestinal parasites.

Sustainable Use of Pesticide Applications in Citrus: A Support Tool for Volume Rate Adjustment.

Rational application of pesticides by properly adjusting the amount of product to the actual needs and specific conditions for application is a key factor for sustainable plant protection. However, current plant protection product (PPP) labels registered for citrus in EU are usually expressed as concentration (%; rate/hl) and/or as the maximum dose of product per unit of ground surface, without taking into account those conditions. In this work, the fundamentals of a support tool, called CitrusVol, developed to recommend mix volume rates in PPP applications in citrus orchards using airblast sprayers, are presented. This tool takes into consideration crop characteristics (geometry, leaf area density), pests, and product and application efficiency, and it is based on scientific data obtained previously regarding the minimum deposit required to achieve maximum efficacy, efficiency of airblast sprayers in citrus orchards, and characterization of the crop. The use of this tool in several commercial orchards allowed a reduction of the volume rate and the PPPs used in comparison with the commonly used by farmers of between 11% and 74%, with an average of 31%, without affecting the efficacy. CitrusVol is freely available on a website and in an app for smartphones.

Spray pesticide applications in Mediterranean citrus orchards: Canopy deposition and off-target losses.

Only a portion of the water volume sprayed is deposited on the target when applying plant protection products with air-assisted axial-fan airblast sprayers in high growing crops. A fraction of the off-target losses deposits on the ground, but droplets also drift away from the site. This work aimed at assessing the spray distribution to different compartments (tree canopy, ground and air) during pesticide applications in a Mediterranean citrus orchard. Standard cone nozzles (Teejet D3 DC35) and venturi drift reducing nozzles (Albuz TVI 80 03) were compared. Applications were performed with a conventional air-assisted sprayer, with a spray volume of around 3000lha-1 in a Navel orange orchard. Brilliant Sulfoflavine (BSF) was used as a tracer. Results showed that only around 46% of the applied spray was deposited on the target trees and around 4% of the spray was deposited on adjacent trees from adjoining rows independently of the nozzle type. Applications with standard nozzles produced more potential airborne spray drift (23%) than those with the drift reducing nozzles (17%) but fewer direct losses to the ground (22% vs. 27%). Indirect losses (sedimenting spray drift) to the ground of adjacent paths were around 7-9% in both cases. The important data set of spray distribution in the different compartments around sprayed orchard (air, ground, vegetation) generated in this work is highly useful as input source of exposure to take into account for the risk assessment in Mediterranean citrus scenario.

Factors influencing the efficacy of two organophosphate insecticides in controlling California red scale, Aonidiella aurantii (Maskell). A basis for reducing spray application volume in Mediterranean conditions.

BACKGROUND: Because society is seeking ways to lessen the environmental impact of agricultural activity, dose adjustment has become a key issue in current plant protection treatments with high spray application volumes, such as on citrus plants. This work investigates, in field conditions, the factors affecting the efficacy of organophosphate insecticides against California red scale (CRS), Aonidiella aurantii (Maskell), when the delivery rate is decreased. Insecticide rate changes were induced by modifying the spray application volumes of two commercial organophosphate pesticides based on chlorpyrifos and chlorpyrifos-methyl. RESULTS: Results showed that, with increase in the spray volume, the coverage and the uniformity of deposition on the canopy increased, but final infestation depended neither on the spray application volume nor on the coverage. Furthermore, final infestation significantly depended on the pest pressure in the plot and the spray volume applied per unit volume of canopy (L m(-3) canopy). Moreover, it was found that the final infestation was influenced by the efficiency of deposition in the applications that were carried out against the second-generation of CRS. CONCLUSION: Because the spray application volume did not affect the final infestation, this research introduces the possibility that reducing the doses of current citrus organophosphate treatments may still allow effective plant protection in Mediterranean conditions.

Chlorpyrifos bioassay and resistance monitoring of San Joaquin Valley California citricola scale populations.

The responses to chlorpyrifos of six populations of citricola scale, Coccus pseudomagnoliarum (Kuwana) (Hemiptera: Coccidae), were tested using a leaf dip bioassay, and two- to nine-fold resistances were found. LC50 responses of nymphs ranged from 7.5 to 68.9 ppm and LC90 responses ranged from 20 to 222 ppm chlorpyrifos. A population tested monthly during August-October showed up to 3.5-fold differences in LC50 responses but no differences in LC90 responses as scale size increased. A diagnostic concentration of 178 ppm chlorpyrifos was used to test 93 populations from throughout the San Joaquin Valley California during 2006-2009 by using a leaf dip bioassay. Of the populations tested, 41% showed > 20% survival after exposure to the diagnostic concentration of chlorpyrifos, indicating resistance problems. Research is needed to relate the level of survival of the scales in the bioassay to the field efficacy of the insecticide. Tulare County citrus growers applied a higher number of organophosphate and carbamate insecticides during the 15-yr period from 1994 to 2008, and these orchards showed a higher average scale survival of chlorpyrifos and a higher number of locations with resistant scale compared with the other San Joaquin Valley counties. Chlorpyrifos resistance is a significant issue for citricola scale management because biological control is ineffective in the San Joaquin Valley and the alternative neonicotinoid and insect growth regulator (IGR) insecticides require more frequent application.

Spray deposition and efficacy of four petroleum-derived oils used against Tetranychus urticae (Acari: Tetranychidae).

Petroleum-derived spray oils (PDSOs) offer an interesting alternative to acaricides to control the twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), a key pest of clementine mandarins, Citrus reticulata Blanco. However, there is a lack of knowledge on how these products should be used. In this study, we evaluated the efficacy of four PDSOs (Sunspray Ultrafine, Volck Miscible, Texaco D-C-Tron Plus, and Agroaceite) at five concentrations (0.5, 1.0, 1.5, 2.0, and 3.0%) against eggs, protonymphs, and adults of T. urticae. We also characterized the PDSOs deposition pattern to find out the possible relationship between this factor and efficacy. In general, for all PDSO assayed, the higher the concentration, the higher the coverage, the mean area of impacts and efficacy on T. urticae. The biggest mean area of the impacts corresponded to Texaco D-C-Tron Plus. This PDSO was the most effective one and its efficacy was independent of concentration for concentrations higher than 1.0%. The same applied for concentrations higher than 1.5-2.0% for Agroaceite, Volck Miscible, and Sunspray Ultrafine, with high efficacies against eggs, protonymphs, and adults. PDSOs are highly effective against T. urticae, the use of these products should be encouraged in integrated citrus pest management programs in Spain. The next step will be to ascertain the efficacy under real field conditions.

Chemical alternatives to malathion for controlling Ceratitis capitata (Diptera: Tephritidae), and their side effects on natural enemies in Spanish citrus orchards.

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is one of the most important fruit pests worldwide. Mediterranean fruit fly control in Spain has been based on organophosphate sprays, especially malathion, mixed with protein baits. However, this insecticide has recently been excluded from annex 1 of the Directive 91/414 CEE, which lists authorized active ingredients for pest control in the European Union. This article reports on the efficacy of four alternative baited insecticides on Mediterranean fruit fly and their side effects on three natural enemies [Cryptolaemus montrouzieri (Mulsant), Neoseiulus californicus (McGregor), and Aphidius colemani (Viereck)] relevant for pest control in citrus agroecosystems. A high Mediterranean fruit fly mortality was obtained for all baited insecticides (phosmet and spinosad) except lambdacyhalothrin, which caused the lowest mortality and showed a novel disabling effect on surviving Mediterranean fruit fly adults. Spinosad proved to be the most selective bait treatment for C. montrouzieri and N. californicus, whereas for A. colemani the most selective bait was phosmet and lambda-cyhalothrin. These findings would contribute to a sustainable chemical control of C. capitata populations under an integrated pest management system in Spanish citrus orchards.

Automatic sex detection of individuals of Ceratitis capitata by means of computer vision in a biofactory.

BACKGROUND: The sterile insect technique (SIT) is acknowledged around the world as an effective method for biological pest control of Ceratitis capitata (Wiedemann). Sterile insects are produced in biofactories where one key issue is the selection of the progenitors that have to transmit specific genetic characteristics. Recombinant individuals must be removed as this colony is renewed. Nowadays, this task is performed manually, in a process that is extremely slow, painstaking and labour intensive, in which the sex of individuals must be identified. The paper explores the possibility of using vision sensors and pattern recognition algorithms for automated detection of recombinants. RESULTS: An automatic system is proposed and tested to inspect individual specimens of C. capitata using machine vision. It includes a backlighting system and image processing algorithms for determining the sex of live flies in five high-resolution images of each insect. The system is capable of identifying the sex of the flies by means of a program that analyses the contour of the abdomen, using fast Fourier transform features, to detect the presence of the ovipositor. Moreover, it can find the characteristic spatulate setae of males. Simulation tests with 1000 insects (5000 images) had 100% success in identifying male flies, with an error rate of 0.6% for female flies. CONCLUSION: This work establishes the basis for building a machine for the automatic detection and removal of recombinant individuals in the selection of progenitors for biofactories, which would have huge benefits for SIT around the globe.