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Abstract Background Tick-borne rickettsioses (TBR) are emerging, neglected, zoonoses, caused by intracellular α-proteobacteria of the genus Rickettsia, that pose a growing public health concern. The aim of the present study was to evaluate rickettsial infections in questing ticks collected from four different ecological areas in mainland Portugal. Methods Over a two-year period, a total of 707 questing ticks were collected. Individual adult ticks and pooled nymphs were submitted to DNA extraction, followed by qPCR assays targeting the gltA rickettsial gene. Positive samples were then submitted to conventional PCR targeting the gltA and the ompA genes for phylogenetic analysis. Results In total, eight tick species were identified: Dermacentor marginatus, Haemaphysalis inermis, Haemaphysalis punctata, Hyalomma lusitanicum, Ixodes frontalis, Ixodes ricinus, Rhipicephalus pusillus, and Rhipicephalus sanguineus sensu lato. Additionally, rickettsial infection was associated with seven of these species, with I. frontalis being the exception. Notably, the prevalence of Rickettsia spp. was 26.35%, with phylogenetic validation confirming infections with R. helvetica, R. massiliae, R. monacensis, Candidatus R. rioja, and R. slovaca. Conclusions The present study highlights the necessity for ongoing surveillance to map and monitor both questing and feeding ticks, along with their vertebrate hosts. Effective control strategies are of utmost importance to mitigate the escalating threat of TBR. Additionally, the present study provides valuable epidemiological insights into TBR in Portugal, including the identification of R. slovaca infecting I. ricinus - an unconventional tick-pathogen relationship - and the first report of Candidatus R. rioja infecting D. marginatus in Portugal. In conclusion, this study contributes with valuable data regarding epidemiological results on ticks and TBR circulating in Portugal, emphasizing the importance of proactive measures to address this emerging public health challenge. Graphical abstract

Moringa oleifera is an Asian tree species cultivated for its nutritional and medicinal properties. Present study was carried out during 2021 and 2022 at Federal Institute of Minas Gerais, São João Evangelista-MG, Doce River Valley, South-Eastern Brazil to determine NPK doses for Moringa and critical NPK levels in the shoot. The experimental design was a fractional factorial of the type (1/2)43, with 32 treatments set up in four blocks. The treatments represented combinations of doses of N (0, 40, 80, 160 kg/ha); P2O5 (0, 45, 90, 180 kg/ha); and K2O (0, 20, 40, 80 kg/ha). Each experimental unit was composed of four seedlings arranged in a row. Plant shoot harvests were carried out every 60 days (January, March, May, July, September and November) for two consecutive years. The data were analyzed using regression analysis, and the adjusted response surface model was: Y = b0 + b1 N + b2 N2 + b3 P + b4 P2 + b5 K + b6 K2 + b7 NP + b8 NK + b9 PK. The productivity of fresh and dry matter of the shoot of M. oleifera plants was influenced only by N doses in the first year of cultivation, when the supply of 88.7 and 85.8 kg/ha of N led to the maximum estimated productivity for fresh (10,360.3 kg/ha) and dry matter (2,84.0 kg/ha). The critical contents in the shoot were 3.23 dag/kg (N), 0.41 dag/kg (P), and 2.18 dag/kg (K), while the annual extraction by plants was 78.44 kg/ ha (N), 8.99 kg/ha (P), and 48.81 kg/ha (K).

Over the years, tick-borne pathogens (TBPs) have garnered significant interest due to their medical, veterinary and economic importance. Additionally, TBPs have drawn attention to how these microorganisms interact with their own vectors, increasing the risk to human and animal infection of emerging and reemerging zoonoses. In this sense, ticks, which are obligate hematophagous ectoparasites, have a key role in maintaining and transmitting TBPs among humans and animals. The aim of this study was to assess the prevalence of neglected TBPs in mainland Portugal, namely Anaplasma spp., Babesia spp., Ehrlichia spp. and Neoehrlichia mikurensis. DNA fragments were detected in questing ticks collected from five different ecological areas under investigation. To the best of the authors’ knowledge, this study reports new worldwide findings, including B. bigemina infecting Ixodes frontalis, Ixodes ricinus and Rhipicephalus sanguineus sensu lato. Additionally, it presents new findings in Portugal of N. mikurensis infecting I. ricinus and of presumably Wolbachia endosymbionts being detected in I. ricinus. Overall, there were 208 tick samples that were negative for all screened TBPs. The results herein obtained raise concerns about the circulation of neglected TBPs in mainland Portugal, especially in anthropophilic ticks, highlighting the importance of adopting a One Health perspective.

Phytoremediation is a technique with high viability due to its low cost and minimal environmental impact. However, there are a few results regarding the use of tropical grasses in the phytoremediation of manganese (Mn), and there is no clear definition of which extractor is more suitable for assessing Mn availability in soils. The objective of this study was to evaluate the growth potential and phytoremediation of five tropical grasses in Mn-contaminated soil, as well as to assess the efficiency of four extractors in Mn extraction. The grass cultivars evaluated included Aruana and Tanzânia of Megathyrsus maximus, Xaraés and Marandu of Urochloa brizantha, and Basilisk of Urochloa decumbens grown in Typic Hapludox (Oxisol) amended with 0, 50, 150, and 450 mg of Mn per kg−1 of soil. The experiment was conducted in a greenhouse, using a completely randomized experimental design in a 5 × 4 factorial scheme, with five replications. The experimental period lasted for 90 days. The dry matter of the shoot and root of the grasses was evaluated. Mn concentration in dry matter and its availability in soil were extracted by Mehlich-1 and DTPA pH 7.3, while the semitotal concentration was determined by USEPA 3051 and total Mn by USEPA 3052 method. Tropical grasses showed reduced growth with increasing Mn rates in the soil, with the following sequence in the reduction in growth: Aruana > Basilisk > Marandu > Xaraés and lastly the Tanzânia. The Aruana cultivar showed greater potential for phytoextraction due to its higher ability to accumulate Mn in its biomass, followed by the Basilisk cultivar. The four different evaluated extractors were efficient in measuring Mn concentrations in the soil, with higher toxicity critical levels for the USEPA 3051 method due to the semitotal extraction nature and USEPA 3052 due to total Mn extraction. The growth of tropical grasses is reduced with increasing Mn rates applied to the soil. The Aruana of M. maximus grass has greater potential for phytoextraction of Mn-contaminated soils, followed by Basilisk of Urochloa decumbens, Xaraés and Marandu of U. brizantha, and Tanzânia of M. maximus. The Mehlich-1, DTPA pH 7.3, USEPA 3051, and 3052 extractors were efficient in extracting and evaluating Mn availability in soil for tropical grasses. The USEPA 3051 and 3052 methods presented higher toxicity critical levels.

Mountain insect biodiversity is unique, but is menaced by different drivers, particularly climate and land-use changes. In mainland Portugal, the highest mountain - Serra da Estrela - is one of the most important biodiversity hotspots, being classified as Natural Park since 1976. Many lepidopteran and odonate species, including rare and protected species, are known to occur in Serra da Estrela, but basic knowledge on their abundance, distribution and ecology is still lacking. Standardised sampling of these communities is crucial to provide valuable biological information to support short-term decision-making for conservation management, setting simultaneously the standards for mountain biodiversity monitoring aiming to tackle the effects of environmental change in the long-term.This study reports novel information on lepidopteran and odonate species diversity, distribution and abundance from Serra da Estrela Natural Park (Portugal). Seventy-two lepidopteran and 26 odonate species were sampled in this protected area, including the first findings of Apatura ilia (Denis & Schiffermüller, 1775), Macromia splendens (Pictet, 1843) and Vanessa virginiensis (Drury, 1773). New populations of Euphydrias aurinia (Rottemburg, 1775) and Oxygastra curtisii (Dale, 1834), protected species under the Habitats Directive, were found in this Natural Park and novel distribution and ecological data were collected for most species, including several rare species and subspecies [e.g. Aeshna juncea (Linnaeus, 1758), Coenonympha glycerion iphioides Staudinger, 1870, Cyaniris semiargus (Rottemburg, 1775) and Sympetrum flaveolum (Linnaeus, 1758)]. All data were collected using standardised sampling allowing its use as a baseline for biodiversity monitoring in Serra da Estrela.

Nickel (Ni) is extremely toxic to plants at high concentrations. Phytoliths have the potential to sequester the heavy metals absorbed by plants and act as a detoxification mechanism for the plant. The authors of the present study aimed to evaluate the effects of Ni on the growth and phytolith yield of grasses in two artificially contaminated soils. Two experiments separated by soil types (Typic Quartzipsamment and Rhodic Hapludox) were conducted in a completely randomized design in a 2 × 4 factorial scheme with three replications. The factors were two species of grass (Urochloa decumbens and Megathyrsus maximus) and three concentrations of Ni (20, 40, and 120 mg kg−1) and control treatment. The grasses were influenced by the increase in Ni rates in the soils. Ni exerted a micronutrient function with the addition of 30 mg kg−1 of Ni in soils, but this concentration caused toxicity in grasses. Such a level is lower than the limits imposed by the Brazilian environmental legislation. Higher Ni availability in Typic Quartzipsamment promoted Ni toxicity, with reduced growth and increased phytolith yield in the shoot, increased Ni in the shoot, and Ni occlusion in phytoliths by grasses, in comparison with Rhodic Hapludox. The yield and Ni capture in phytoliths by grasses in Ni-contaminated soils are related to the genetic and physiological differences between grasses and Ni availability in soils. Ni capture by phytoliths indicates that it may be one of the detoxification mechanisms of Urochloa decumbens to Ni contamination, providing additional tolerance. Megathyrsus maximus may be a future grass for the phytoremediation technique in Ni-contaminated soils.

The control of ticks through vaccination offers a sustainable alternative to the use of chemicals that cause contamination and the selection of resistant tick strains. However, only a limited number of anti-tick vaccines have reached commercial realization. In this sense, an antigen effective against different tick species is a desirable target for developing such vaccines. A peptide derived from the tick P0 protein (pP0) conjugated to a carrier protein has been demonstrated to be effective against the Rhipicephalus microplus, Rhipicephalus sanguineus, and Amblyomma mixtum tick species. The aim of this work was to assess the efficacy of this peptide when conjugated to the Bm86 protein against Dermacentor nitens and Ixodes ricinus ticks. An RNAi experiment using P0 dsRNA from I. ricinus showed a dramatic reduction in the feeding of injected female ticks on guinea pigs. In the follow-up vaccination experiments, rabbits were immunized with the pP0-Bm86 conjugate and challenged simultaneously with larvae, nymphs, and the adults of I. ricinus ticks. In the same way, horses were immunized with the pP0-Bm86 conjugate and challenged with D. nitens larva. The pP0-Bm86 conjugate showed efficacies of 63% and 55% against I. ricinus and D. nitens ticks, respectively. These results, combined with previous reports of efficacy for this conjugate, show the promising potential for its development as a broad-spectrum anti-tick vaccine.

Prior research found an association between mother–infant attachment and antibiotic use. Ambivalent-attached infants are more likely to take antibiotics than other infants, and their mothers tend to be less sensitive to their needs than most. This finding is important because it shows the association between psychological processes, early relationships, and health outcomes. We aim to learn about children with high-risk attachment relationships, such as disorganized-attached infants. This study compares antibiotic use, infant–mother interactive behavior, and health indicators according to infant attachment patterns (including disorganized attachment). For this purpose, we observed mothers–infants’ interactive behavior in free play at nine months and infants’ attachment in the Ainsworth Strange Situation at twelve months. Participants included 77 girls and 104 boys (full-term and preterm) and their mothers. Paradoxically, mothers of disorganized-attached infants reported that their children were ill only 1.56 times on average, but 61% of their children used antibiotics in the first nine months. The other mothers reported that their children were sick 5.73 times on average, but only 54% of their children used antibiotics in the same period. Infants with disorganized attachment had mothers who were more literate and less sensitive. These results add to a body of research that shows that early high-risk relationships affect children’s lives at multiple levels.

Antibiotics have individual and public-health drawbacks. Nevertheless, mother-infant attachment quality and maternal sensitivity are associated with antibiotic use. Ambivalent-attached infants are more likely to consume antibiotics than other infants. Conceivably, the emotional over-externalization of ambivalent-attached infants and maternal anxiety when infants are ill raise concerns in healthcare professionals, leading to antibiotic over-prescriptions. However, because infants prematurely born, particularly those with less than 32 weeks of gestation, are under more accurate health vigilance, the impact of infant and maternal behavior on antibiotic prescription may vanish in this sample. To test this hypothesis, we performed a longitudinal study to compare antibiotic use and the quality of mother-infant attachment in three groups: 86 infants born at full-term, 44 moderate-to-late preterm infants (32–36 gestation weeks), and 58 very-to-extreme preterm infants (

Neoehrlichia mikurensis is a new emerging tick-borne Gram-negative bacterium, belonging to the family Anaplasmataceae, the main vector of which in Europe is the tick Ixodes ricinus. N. mikurensis is responsible for neoehrlichiosis, occurring mostly in patients with underlying diseases. In the present study, a total of 348 I. ricinus and Dermacentor reticulatus ticks collected in north-eastern Poland were analyzed for the prevalence of N. mikurensis. A total of 140 questing ticks (124 of I. ricinus ticks and 16 D. reticulatus) collected with the flagging method and 208 ticks (105 and 103 I. ricinus and D. reticulatus, respectively) removed from dogs were selected for the study. cDNA (questing ticks) and total DNA (questing and feeding ticks) were analyzed by qPCR targeting the 16S rRNA gene of N. mikurensis. Positive samples were further analyzed by nested PCR and sequencing. The prevalence differed between ticks collected from vegetation (19.3%; 27/140) and ticks removed from dogs (6.7%; 14/208). The presence of the pathogen in questing and feeding D. reticulatus ticks was proven in Poland for the first time. In summary, our research showed that infections of ticks of both the most common tick species I. ricinus and D. reticulatus in north-eastern Poland are present and ticks collected from urban areas were more often infected than ticks from suburban and natural areas. The detection of N. mikurensis in I. ricinus and D. reticulatus ticks from north-eastern Poland indicates potential transmission risk for tick-bitten humans at this latitude.

Mountain ecosystems are important biodiversity hotspots and valuable natural laboratories to study community assembly processes. Here, we analyze the diversity patterns of butterflies and odonates in a mountainous area of high conservation value—Serra da Estrela Natural Park (Portugal)—and we assess the drivers of community change for each of the two insect groups. The butterflies and odonates were sampled along 150 m transects near the margins of three mountain streams, at three elevation levels (500, 1000, and 1500 m). We found no significant differences in odonate species richness between elevations, but marginal differences (p = 0.058) were found for butterflies due to the lower number of species at high altitudes. Both insect groups showed significant differences in beta diversity (βtotal) between elevations, with species richness differences being the most important component for odonates (βrich = 55.2%), while species replacement drove the changes between butterfly assemblages (βrepl = 60.3%). Climatic factors, particularly those depicting harsher conditions of temperature and precipitation, were the best predictors of total beta diversity (βtotal) and its components (βrich, βrepl) for the two study groups. The study of insect biodiversity patterns in mountain ecosystems and of the role played by different predictors contribute to further our understanding on the community assembly processes and may help to better predict environmental change impacts on mountain biodiversity.

Anti-tick vaccines development mainly depends on the identification of suitable antigens, which ideally should have different features. These should be key molecules in tick biology, encoded by a single gene, expressed across life stages and tick tissues, capable of inducing B and T cells to promote an immunological response without allergenic, hemolytic, and toxic effects; and should not be homologous to the mammalian host. The discussion regarding this subject and the usefulness of “exposed” and “concealed” antigens was effectively explored in the publication by Nuttall et al. (2006). The present commentary intends to debate the relevance of such study in the field of tick immunological control.

Rhipicephalus microplus is the only tick species known to serve as a biological vector of Theileria equi for horses and other equids in Brazil. The protozoan T. equi is one of the causal agents of equine piroplasmosis, a major threat in horse breeding systems. Vector competence is closely linked to the pathogens’ ability to evade tick defense mechanisms. However, knowledge of tick immune response against infections by hemoparasites of the Theileria genus is scarce. In the present study, the expression of genes involved in immune signaling pathways of R. microplus adults’ guts when challenged with a high or low parasitic load of T. equi was evaluated. This research demonstrates divergences in the immune gene expression pattern linked to T. equi infection in R. microplus since the Toll, IMD, and JNK signaling pathways were transcriptionally repressed in the guts of adult ticks infected with T. equi. Moreover, the results showed that different infectious doses of T. equi induce differential gene expression of key components of immune signaling cascades in R. microplus gut, suggesting a link between the intensity of infection and the activation of tick immunity response. The present study adds knowledge to elucidate the gut immune signaling response of R. microplus to T. equi infection. In addition, the generated data can serve as a basis for further investigations to develop strategies for controlling and preventing equine piroplasmosis.

Tick-borne rickettsioses (TBR) are caused by obligate, intracellular bacteria of the spotted-fever group (SFG) of the genus Rickettsia (Order Rickettsiales), transmitted by hard ticks. TBR are one of the oldest known vector-borne zoonoses and pose a threat to both human and animal health, as over the years, new SFG Rickettsia spp. have been reported worldwide with the potential to be human pathogens. In Portugal and Spain, the countries that constitute the Iberian Peninsula, reported TB rickettsiae causing human disease include Rickettsia conorii conorii, Rickettsia conorii israelensis, Rickettsia slovaca, Rickettsia raoultii, Candidatus Rickettsia rioja, Rickettsia sibirica mongolitimonae, and Rickettsia monacensis. An allochthonous case of TBR caused by Rickettsia massiliae, described in Spain, points to the need to monitor disease epidemiology, to predict risks of exposure and spread of disease, and taking into account globalization and climate changes. This review aims to provide up-to-date information on the status of TBR in the Iberian Peninsula, as well as to show the importance of a national and international collaborative epidemiology surveillance network, towards monitoring Rickettsia spp. circulation in both Portugal and Spain.

Bartonella spp. comprises emergent and re-emergent fastidious Gram-negative bacteria with worldwide distribution. Cats are the main reservoir hosts for Bartonella henselae and dogs represent opportunistic hosts for the bacteria. Even though ticks may also play a role in transmission, their competence as vectors for Bartonella spp. has not been totally understood. Considering only a few studies had a focus on screening Bartonella in animals, humans and ectoparasites in Portugal, this study aimed to address the molecular occurrence of Bartonella sp. in 123 stray cats, 25 stray dogs, 30 humans from Lisbon and 236 questing ticks within the country. Using a qPCR targeting the nuoG gene, it was possible to detect Bartonella sp. DNA on 20.32% of cat samples (25/123). From these positive samples, 13 sequences were characterized as B. henselae, 11 as B. clarridgeiae and 1 presented co-infection with both species. The absolute quantification of nuoGBartonella DNA in sampled cats ranged from 2.78 × 10 to 1.03 × 105 copies/µL. The sampled dogs, humans and ticks were negative. These results showed that B. henselae and B. clarridgeiae are circulating in stray cats from Lisbon. Additional and more extended studies should be conducted to determine the impact of such infections on humans, particularly those in constant and direct contact with cats.

Phytoremediation is a technique with high viability due to its low cost and minimal environmental impact. However, there are a few results regarding the use of tropical grasses in the phytoremediation of manganese (Mn), and there is no clear definition of which extractor is more suitable for assessing Mn availability in soils. The objective of this study was to evaluate the growth potential and phytoremediation of five tropical grasses in Mn‐contaminated soil, as well as to assess the efficiency of four extractors in Mn extraction. The grass cultivars evaluated included Aruana and Tanzânia of Megathyrsus maximus, Xaraés and Marandu of Urochloa brizantha, and Basilisk of Urochloa decumbens grown in Typic Hapludox (Oxisol) amended with 0, 50, 150, and 450 mg of Mn per kg−1 of soil. The experiment was conducted in a greenhouse, using a completely randomized experimental design in a 5 × 4 factorial scheme, with five replications. The experimental period lasted for 90 days. The dry matter of the shoot and root of the grasses was evaluated. Mn concentration in dry matter and its availability in soil were extracted by Mehlich‐1 and DTPA pH 7.3, while the semitotal concentration was determined by USEPA 3051 and total Mn by USEPA 3052 method. Tropical grasses showed reduced growth with increasing Mn rates in the soil, with the following sequence in the reduction in growth: Aruana > Basilisk > Marandu > Xaraés and lastly the Tanzânia. The Aruana cultivar showed greater potential for phytoextraction due to its higher ability to accumulate Mn in its biomass, followed by the Basilisk cultivar. The four different evaluated extractors were efficient in measuring Mn concentrations in the soil, with higher toxicity critical levels for the USEPA 3051 method due to the semitotal extraction nature and USEPA 3052 due to total Mn extraction. The growth of tropical grasses is reduced with increasing Mn rates applied to the soil. The Aruana of M. maximus grass has greater potential for phytoextraction of Mn‐contaminated soils, followed by Basilisk of Urochloa decumbens, Xaraés and Marandu of U. brizantha, and Tanzânia of M. maximus. The Mehlich‐1, DTPA pH 7.3, USEPA 3051, and 3052 extractors were efficient in extracting and evaluating Mn availability in soil for tropical grasses. The USEPA 3051 and 3052 methods presented higher toxicity critical levels. [ABSTRACT FROM AUTHOR]

Nickel (Ni) is extremely toxic to plants at high concentrations. Phytoliths have the potential to sequester the heavy metals absorbed by plants and act as a detoxification mechanism for the plant. The authors of the present study aimed to evaluate the effects of Ni on the growth and phytolith yield of grasses in two artificially contaminated soils. Two experiments separated by soil types (Typic Quartzipsamment and Rhodic Hapludox) were conducted in a completely randomized design in a 2 × 4 factorial scheme with three replications. The factors were two species of grass (Urochloa decumbens and Megathyrsus maximus) and three concentrations of Ni (20, 40, and 120 mg kg−1) and control treatment. The grasses were influenced by the increase in Ni rates in the soils. Ni exerted a micronutrient function with the addition of 30 mg kg−1 of Ni in soils, but this concentration caused toxicity in grasses. Such a level is lower than the limits imposed by the Brazilian environmental legislation. Higher Ni availability in Typic Quartzipsamment promoted Ni toxicity, with reduced growth and increased phytolith yield in the shoot, increased Ni in the shoot, and Ni occlusion in phytoliths by grasses, in comparison with Rhodic Hapludox. The yield and Ni capture in phytoliths by grasses in Ni-contaminated soils are related to the genetic and physiological differences between grasses and Ni availability in soils. Ni capture by phytoliths indicates that it may be one of the detoxification mechanisms of Urochloa decumbens to Ni contamination, providing additional tolerance. Megathyrsus maximus may be a future grass for the phytoremediation technique in Ni-contaminated soils. [ABSTRACT FROM AUTHOR]