Impact of different Meloidogyne species on the development of sugarcane plants.

This study aimed to evaluate the impact of Meloidogyne javanica, Meloidogyne incognita, and Meloidogyne arenaria on different aspects of the development of sugarcane plants under greenhouse conditions. For this purpose, seedlings of the RB867515 genotype were individually inoculated with 5,000 eggs + second-stage juveniles of their respective nematodes/plant, and non-inoculated plants were used as control. After 330 days of inoculation, the plants were removed from the pots, and the following characteristics were evaluated: fresh mass of the aerial part and root system; leaf area; leaf chlorophyll index; culm diameter; fresh mass of culms; broth volume; contents of neutral and acid detergent fiber, cellulose, hemicellulose, lignin, apparent sucrose in broth, and reducing sugars in broth; total soluble solids concentration. Subsequently, the final population of nematodes in the root system of inoculated plants was determined to calculate the reproduction factor of nematodes. The results showed that all tested Meloidogyne species negatively affected plant development and the composition of some analyzed fractions, in comparison to the non-inoculated control. However, the presence of the root-knot nematode in sugarcane plants increased the contents of neutral and acid detergent fiber, cellulose, hemicellulose, lignin, and reducing sugars, regardless of the Meloidogyne species.

Oil reaching the coast: Is Brazil on the route of international oceanic dumping?

After the oil spill disaster occurred in 2019, various events of tar balls reaching the Brazilian coast and archipelagos have been reported. The hypothesis here is that the oil/waste dumped in international waters by ships on-route to Cape of Good Hope is reaching the Brazilian coast. On that account, 30-year probabilistic simulations were used to estimate the probability of dumped oil residue reaching the Brazilian coast. The simulations considered three Zones following the South Atlantic route. The results have shown that up to 28.5 % of large ships could dump oil on-route. Inside the Brazilian Exclusive Economic Zone, the probability of dumped oil/waste reaching the coastline is about 62 % and quickly decreases for Dumping Zones 2 and 3. Equatorial and Northeast shores of Brazil are the most vulnerable to oceanic dumping when compared to other regions.

[Evaluation of the Manchester Triage System in Patients with Acute Primary Angle Closure Attack: A Retrospective Study]. / Avaliação do Sistema de Triagem de Manchester em Doentes com Crise de Encerramento Agudo Primário do Ângulo Iridocorneano: Um Estudo Retrospetivo.

INTRODUCTION: Acute primary angle closure attack is an ophthalmological emergency. The aim of this study was to describe the cases diagnosed in the Emergency Department, by correlating the initial complaint with the Manchester triage level and ultimately the time needed until ophthalmological evaluation and iridotomy. MATERIAL AND METHODS: Retrospective analysis of the electronic medical records of patients with acute primary angle closure attack that attended the Ophthalmology Emergency Department of our tertiary center between January 2010 and December 2020. Overall, 2228 Emergency Department episodes coded with the diagnoses glaucoma or ocular hypertension were retrieved, followed by screening of each episode for correct identification of true acute primary angle closure attacks. Clinical data was gathered, including Manchester triage level, presenting complaint, intraocular pressure at presentation, first medical specialty that observed the patient, time until observation by Ophthalmology and time until laser iridotomy. RESULTS: Among the 120 patients identified, 84 (70%) were female and the mean age was 68 ± 12 years. Mean intraocular pressure at admission was 53.4 ± 12.4 mmHg, and 9.2% of patients presented only non-ocular complaints, while 9.2% presented mixed complaints (ocular and non-ocular). Most patients (68.1%) with only non-ocular or mixed complaints were triaged to a non-ophthalmologist (p < 0.001). Concerning the triage system, at admission, most patients (66.7%) were labelled yellow (urgent), while 9.2% and none were labelled as orange (very urgent) or red (emergent), respectively. Most patients (83.3%) were directly sent to Ophthalmology (properly triaged), while the remaining were incorrectly assigned to a non-ophthalmologist. Median time until observation by Ophthalmology was 49 minutes in the properly triaged group (min. 15, max. 404), while it was 288 minutes (min. 45, max. 871) in those who were incorrectly triaged (p < 0.001). Likewise, median time until treatment with laser iridotomy was 203 minutes in the properly triaged group (min. 22, max. 1440) and 353 minutes in the incorrectly triaged group (min.112, max. 947) (p < 0.001). CONCLUSION: Most patients with acute primary angle closure attack were not properly triaged according to the level of the Manchester triage system. There was a significant delay in the diagnosis and treatment of those patients who were first assigned to non-ophthalmologists. There is a need to raise awareness regarding the presenting signs and symptoms of an acute primary angle closure attack in order to avoid preventable vision loss.

Introdução: A crise de encerramento agudo primário do ângulo iridocorneano é uma emergência oftalmológica. O objetivo deste estudo foi descrever os casos admitidos no Serviço de Urgência do Centro Hospitalar Universitário São João, correlacionando a queixa inicial com o nível de triagem de Manchester atribuído e o tempo até observação por Oftalmologia e realização de iridotomia. Material e Métodos: Análise retrospetiva dos registos clínicos dos doentes com encerramento agudo primário do ângulo, admitidos no Serviço de Urgência entre janeiro de 2010 e dezembro de 2020. Foram revistos 2228 episódios com diagnóstico de glaucoma ou hipertensão ocular para identificação correta dos casos de crise de encerramento do ângulo. Foram extraídas variáveis, nomeadamente o nível de triagem de Manchester atribuído, queixa principal, pressão intraocular à admissão, especialidade responsável pelo primeiro contacto médico e tempos até observação por Oftalmologia e até iridotomia. Resultados: Foram identificados 120 doentes, 84 (70%) do sexo feminino, com idade média de 68 ± 12 (desvio padrão) anos. A pressão intraocular média à admissão foi de 53,4 ± 12,4 mmHg. Em 9,2% dos doentes a queixa principal foi não-ocular, enquanto 9,2% apresentavam queixas não-oculares e oculares associadas. A maioria (68,1%) dos doentes com queixas não-oculares ou mistas foi triada para um não-oftalmologista. Segundo o sistema de triagem, a maioria (66,7%) dos doentes foi triada com nível amarelo (urgente), 9,2% foram triados com laranja (muito urgente) e nenhum vermelho (emergente). O primeiro especialista a observar os doentes após a triagem foi um oftalmologista em 83,3% dos casos (corretamente triados), enquanto os restantes foram inicialmente observados por outra especialidade. O tempo mediano até observação por Oftalmologia foi de 288 minutos (min. 45, máx. 871) num doente incorretamente triado e 49 minutos (min. 15, máx. 404) (p < 0,001) em doentes corretamente triados. O tempo mediano até realização de iridotomia laser foi de 353 minutos (min. 112, máx. 947) nos doentes incorretamente triados e 203 minutos (min. 22, máx. 1440) nos corretamente triados (p < 0,001). Conclusão: A maioria dos doentes com crise de encerramento agudo primário do ângulo iridocorneano não foi triada de acordo com o grau de prioridade apropriado segundo o sistema de triagem de Manchester. Nos doentes que não foram imediatamente seguidos por Oftalmologia verificou-se um atraso significativo no diagnóstico e início do tratamento. Torna-se premente a consciencialização dos profissionais de saúde sobre esta condição clínica e a otimização do processo de triagem para minimizar a perda de visão.

Production of Protein Concentrates from Macauba (Acrocomia aculeata and Acrocomia totai) Kernels by Sieve Fractionation.

Macauba palm fruits (Acrocomia aculeata and Acrocomia totai) are emerging as sources of high-quality oils from their pulp and kernels. The protein-rich macauba kernel meal (MKM) left after oil extraction remains undervalued, mainly due to the lack of suitable deoiling parameters and integrated protein recovery methods. Therefore, the present study aimed to produce protein concentrates from MKM using sieve fractionation. The deoiling parameters, comprising pressing, milling, and solvent extraction, were improved in terms of MKM functionality. The combination of hydraulic pressing, milling to 1 mm, and the hexane extraction of A. aculeata kernels resulted in MKM with the highest protein solubility (77.1%), emulsifying activity index (181 m2/g protein), and emulsion stability (149 min). After sieve fractionation (cut size of 62 µm), this meal yielded a protein concentrate with a protein content of 65.6%, representing a 74.1% protein enrichment compared to the initial MKM. This protein concentrate showed a reduced gelling concentration from 8 to 6%, and an increased emulsion stability from 149 to 345 min, in comparison to the MKM before sieving. Therefore, sieve fractionation after improved deoiling allows for the simple, cheap, and environmentally friendly recovery of MKM proteins, highlighting the potential of macauba kernels as a new source of protein.

Afghan children and adolescents: The burden of poor mental health in contexts of widespread poverty, social inequality and persistent violence.

In Afghanistan, the burden of poor mental health arises in contexts of widespread poverty, social inequality, and persistent violence. Generations of Afghans were born during the conflict, and some never knew peace. Children make up more than half (57%) of the population in need of emergency humanitarian assistance in Afghanistan. Recent assessments indicate that more than a third of children have been exposed to psychological distress due to the loss of family and community members and the constant risk of death and injury. Children and teens are struggling with anxieties and fears, desperately in need of mental health support. The lack of social support manifested itself in various forms of suffering in both children and adolescents. The quality of the environment where children and adolescents grow up shapes their well-being and development.

Echoes of Global Silence: Is there protection and safety for Venezuelan children and adolescents?

As the crisis in Venezuela deepens, an increasing number of children urgently needs shelter, protection, and access to basic services, including food, medicine, clean water, and sanitation. Children and young people in transit are particularly at risk of criminal activity or being separated from their families. The consequences of the humanitarian crisis for children could be devastating for the country's future. The child labor problem was fueled by a mass migration of more than five million Venezuelans that turned many children into livelihoods for their families. The pandemic has aggravated risk factors for child labor. The work ranges from working in dumps to agricultural fields, adding that children in rural areas are more likely to depend on public assistance and are at greater risk of being recruited by gangs. Some Venezuelan women and girls are traveling for hours or days to cross the Colombian border and earn money as sex workers. The complex and multifaceted reality of international migration reveals enormous challenges that directly affect the lives of children and adolescents, especially the most vulnerable, and demand urgent responses from the constituted powers and civil society in the face of countless human rights violations those people experience.

First description of the advertisement call of the rare treefrog Aplastodiscus flumineus (Cruz and Peixoto, 1985) (Anura: Hylidae: Cophomantinae).

N/A.

Rediscovery of the toadlet Brachycephalus bufonoides Miranda-Ribeiro, 1920 (Anura: Brachycephalidae) with osteological and acoustic descriptions.

Brachycephalus bufonoides was described as a "variety" of B. ephippium based on two specimens which 90 years later was considered full species. Besides its brief original description, nothing else is known for this species. Herein we report the rediscovery of the pumpkin-toadlet Brachycephalus bufonoides from Nova Friburgo, State of Rio de Janeiro, the second most populous area within the Atlantic Forest in Brazil. A detailed osteological description of this species was also provided, including skull, hyolaryngeal skeleton and postcranium skeleton. The laryngeal skeleton of Brachycephalus genus was depicted for the first time. We conducted a molecular phylogenetic analysis of Brachycephalus using DNA sequences comprising two fragments of mitochondrial gene (16S). Both analysis with Bayesian inference and maximum parsimony supported the recognition of B. bufonoides as an exclusive lineage, allocated within the B. ephippium species group in B. vertebralis lineage. We improved the diagnosis and variation of the species, including more collected specimens, coloration in vivo and advertisement call description. Compared with its congeners, B. bufonoides has skin on head and dorsum with dermal hyperossification; skull with hyperossification of postorbital crests; a pair of hyperossified bulges about equidistant between postorbital crests; fourth presacral vertebra with transverse process hyperossified, ornamented and sacral diapophyses hyperossified, which can be seen externally (lineage of B. vertebralis sensu Condez et al. 2020); presence of dermal ossification as separated bulges of each vertebrae; general background color orange with different intensities of dark orange blotches on dorsum, including bordering of sacral region; absence of osteoderms and presence of warts on the dorsolateral surface of body; medium body size (SVL of adults: 12.0-14.5 mm for males and 14.7-16.3 mm for females; Table 1); rough dorsum; advertisement calls with 13 to 17 pulses; presence of pulse period modulation; and advertisement calls with notes longer than 0.2 s (0.22 to 0.31 s). Herein an important contribution for the taxonomy and systematics of this genus is provided, including a large amount of novel information for B. bufonoides from different sources (i.e., molecular, morphological variation, bioacoustic), allowing it to be included in future studies of species delimitation and relationships within Brachycephalus. Also, the discovery of this species reiterates the importance of Nova Friburgo for the conservation of the Atlantic Forest biodiversity.

Parque Nacional da Serra dos Órgãos: the highest Amphibian diversity within an Atlantic Forest protected area / Parque Nacional da Serra dos Órgãos: a maior diversidade de anfíbios em uma área de proteção da Mata Atlântica

Abstract: We studied the amphibian community of the Parque Nacional da Serra dos Órgãos (PARNASO) for over thirty years. The area of 20,024 hectares has a steep altitudinal gradient (200-2,263 m a.s.l.), and it is located in the municipalities of Guapimirim, Magé, Petrópolis and Teresópolis, middle of the state of Rio de Janeiro, Brazil. Most data were obtained from sampling sites in the municipality of Teresópolis, with additional data from zoological collections and bibliography. We recorded 83 amphibian species distributed in two orders, Anura, 13 families: Aromobatidae (1), Brachycephalidae (11), Bufonidae (5), Centrolenidae (2), Craugastoridae (2), Cycloramphidae (8), Hemiphractidae (7), Hylidae (28), Hylodidae (6), Leptodactylidae (5), Microhylidae (1), Odontophrynidae (3), Phyllomedusidae (3) and Gymnophiona, one family: Siphonopidae (1). In addition, we present six species that occurs in the buffer zone. Ten of these species are endemic of the park, 18 have PARNASO as its type locality, and five the type locality is at the buffer zone.

Resumo: Estudamos a comunidade de anfíbios do Parque Nacional da Serra dos Órgãos (PARNASO) por mais de 30 anos. A área de 20.024 hectares possui um gradiente altitudinal de 200 a 2.263m acima do nível do mar, e está localizada nos municípios de Guapimirim, Magé, Petrópolis e Teresópolis, que ficam no meio do estado do Rio de Janeiro, Brasil. A maioria das coletas foram feitas no município de Teresópolis, com dados adicionais de outras coleções zoológicas e de bibliografia. Foram registradas ao todo 83 espécies de anfíbios distribuídos em duas ordens, Anura, 13 famílias: Aromobatidae (1), Brachycephalidae (11), Bufonidae (5), Centrolenidae (2), Craugastoridae (2), Cycloramphidae (8), Hemiphractidae (7), Hylidae (28), Hylodidae (6), Leptodactylidae (5), Microhylidae (1), Odontophrynidae (3), Phyllomedusidae (3) e Gymnophiona, uma família: Siphonopidae (1). Foram registradas, também, seis espécies que ocorrem na zona de amortecimento. Destas espécies 10 são endêmicas, 18 têm o PARNASO como localidade tipo e cinco têm a localidade tipo na zona de amortecimento.

Calls of Boanalatistriata (Caramaschi & Cruz, 2004) (Amphibia, Anura, Hylidae), an endemic tree frog from the State of Minas Gerais, Brazil.

Bioacoustical data are useful for studying amphibians, especially their conservation, taxonomy, and evolution, among others. Of the 12 species of the Boanapolytaenia clade, only B.buriti and B.latistriata have no published information about their advertisement calls. We recorded four males of B.latistriata in its type locality at Parque Nacional do Itatiaia, south-eastern Brazil. We used a Roland R26 digital recorder with a Sennheiser ME-67 microphone and analysed the recordings using the Raven Pro 1.5 software. We recorded two different types of calls (call A and call B). Both were composed of one pulsed note and presented a slightly ascending-descending frequency modulation. Call A was more frequent, having durations between 0.042 and 0.093 s with the dominant frequency ranging from 3375.0 to 3937.5 Hz, and was composed of 11 to 21 pulses separated by intervals that were not fully silent. Call B had durations between 0.711 and 1.610 s, with dominant frequency from 3281.2 to 3750.0 Hz, and was composed of 11 to 29 pulses separated by fully silent intervals. Among the B.polytaenia clade, the calls of B.latistriata are more similar to those of B.bandeirantes, B.beckeri, B.polytaenia, and B.aff.beckeri. The calls of B.latistriata differ from these species in its lower dominant frequency. Boanalatistriata present a short, single-note call with a lower pulse period (call A) and a long call with higher pulse period (call B), which differ from the other species of the clade. The coefficients of variation for the various bioacoustical attributes were calculated within- and between-males and these have been discussed. We also report a fight event between two males of B.latistriata. This is the first report of a fight in members of the B.polytaenia clade.

Amphibian chytrid infection is influenced by rainfall seasonality and water availability.

Amphibians suffer from a number of factors that make them the most threatened group of vertebrates. One threat is the fungal disease chytridiomycosis caused by the emerging pathogen Batrachochytrium dendrobatidis (Bd), which has rapidly spread and caused the loss of massive amphibian biodiversity worldwide. Recently, Bd was associated with a few amphibian population declines and extinctions in some areas of the Brazilian Atlantic Forest. However, the mechanisms underlying such declines are not fully understood. Therefore, it is essential to improve our knowledge of abiotic factors that can possibly influence Bd prevalence and chytridiomycosis disease severity. Herein we tested the hypothesis that water availability (such as in perennial streams, where Bd is frequently present in larvae) and rainfall would increase the prevalence of Bd. To test this, we sampled frogs from 6 transects with different numbers of perennial waterbodies, and we report that the more water available in the area, the higher the probability of Bd infection on anurans. Seasonality also influenced both the Bd prevalence in the area and the intensity of infection in infected frogs. However, Bd prevalence was higher during the rainy months whereas the infection burden was lower. We suggest that Bd is likely spread during the summer, when most anuran species gather near the water for spawning and when rainfall overfills ephemeral wetlands. On the other hand, during the drier months, a higher infection burden may be explained by increased disease susceptibility.

Chemical pretreatment of Arundo donax L. for second-generation ethanol production

Background: Pretreatment of lignocellulosic biomass is essential for using it as a raw material for chemical and biofuel production. This study evaluates the effects of variables in the chemical pretreatment of the Arundo biomass on the glucose and xylose concentrations in the final enzymatic hydrolysate. Three pretreatments were tested: acid pretreatment, acid pretreatment followed by alkaline pretreatment, and alkaline pretreatment. Results: The amounts of glucose and xylose released by the enzymatic hydrolysis of the Arundo biomass obtained from acid pretreatment ranged from 6.2 to 19.1 g/L and 1.8 to 3.1 g/L, respectively. The addition of alkaline pretreatment led to a higher yield from the enzymatic hydrolysis, with the average glucose concentration 3.5 times that obtained after biomass hydrolysis with an acid pretreatment exclusively. The use of an alkaline pretreatment alone resulted in glucose and xylose concentrations similar to those obtained in the two-step pretreatment: acid pretreatment followed by alkaline pretreatment. There was no significant difference in 5-hydroxymethylfurfural, furfural, or acetic acid concentrations among the pretreatments. Conclusion: Alkaline pretreatment was essential for obtaining high concentrations of glucose and xylose. The application of an alkaline pretreatment alone resulted in high glucose and xylose concentrations. This result is very significant as it allows a cost reduction by eliminating one step.

A fifth species of the genus Euparkerella (Griffths, 1959), the advertisement calls of E. robusta Izecksohn, 1988 and E. tridactyla Izecksohn, 1988, and a key for the Euparkerella species (Anura: Brachycephaloidea: Craugastoridae).

A new species of the anuran genus Euparkerella is described from a rainforest area in the state of Rio de Janeiro, southeastern Brazil. Morphologically, the species resembles E. brasiliensis and E. cochranae, but differs from them in acoustic features. Relative to its congeners, the new species is characterized by: (1) medium size; (2) slender body; (3) narrow head; (4) long Finger IV, Toes I and V; (5) tubercles of the hand and foot protuberant; (6) duration of advertisement call longer than three seconds; (7) pulse-section rate slower than two sections/second; and (8) exhibiting pulse clusters. The advertisement calls of E. robusta and E. tridactyla are described and a key based on morphological and acoustic characters is presented for species in the genus.

Seasonal Variation in Population Abundance and Chytrid Infection in Stream-Dwelling Frogs of the Brazilian Atlantic Forest.

Enigmatic amphibian declines were first reported in southern and southeastern Brazil in the late 1980s and included several species of stream-dwelling anurans (families Hylodidae and Cycloramphidae). At that time, we were unaware of the amphibian-killing fungus Batrachochytrium dendrobatidis (Bd); therefore, pollution, habitat loss, fragmentation and unusual climatic events were hypothesized as primary causes of these declines. We now know that multiple lineages of Bd have infected amphibians of the Brazilian Atlantic forest for over a century, yet declines have not been associated specifically with Bd outbreaks. Because stream-dwelling anurans occupy an environmental hotspot ideal for disease transmission, we investigated temporal variation in population and infection dynamics of three stream-adapted species (Hylodes asper, H. phyllodes, and Cycloramphus boraceiensis) on the northern coast of São Paulo state, Brazil. We surveyed standardized transects along streams for four years, and show that fluctuations in the number of frogs correlate with specific climatic variables that also increase the likelihood of Bd infections. In addition, we found that Bd infection probability in C. boraceiensis, a nocturnal species, was significantly higher than in Hylodes spp., which are diurnal, suggesting that the nocturnal activity may either facilitate Bd zoospore transmission or increase susceptibility of hosts. Our findings indicate that, despite long-term persistence of Bd in Brazil, some hosts persist with seasonally variable infections, and thus future persistence in the face of climate change will depend on the relative effect of those changes on frog recruitment and pathogen proliferation.

Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses.

The process of selection and validation of reference genes is the first step in studies of gene expression by real-time quantitative polymerase chain reaction (RT-qPCR). The genome of lettuce, the most popular leaf vegetable cultivated worldwide, has recently been sequenced; therefore, suitable reference genes for reliable results in RT-qPCR analyses are required. In the present study, 17 candidate reference genes were selected, and their expression stability in lettuce leaves under drought, salt, heavy metal, and UV-C irradiation conditions and under the application of abscisic acid (ABA) was evaluated using geNorm and NormFinder software. The candidate reference genes included protein-coding traditional and novel reference genes and microRNAs (miRNAs). The results indicate that the expression stability is dependent on the experimental conditions. The novel protein-coding reference genes were more suitable than the traditional reference genes under drought, UV-C irradiation, and heavy metal conditions and under the application of ABA. Only under salinity conditions were the traditional protein-coding reference genes more stable than the novel genes. In addition, the miRNAs, mainly MIR169, MIR171/170 and MIR172, were stably expressed under the abiotic stresses evaluated, representing a suitable alternative approach for gene expression data normalization. The expression of phenylalanine ammonia lyase (PAL) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) was used to further confirm the validated protein-coding reference genes, and the expression of MIR172 and MIR398 was used to confirm the validated miRNA genes, showing that the use of an inappropriate reference gene induces erroneous results. This work is the first survey of the stability of reference genes in lettuce and provides guidelines to obtain more accurate RT-qPCR results in lettuce studies.

Isolation of high-quality RNA from grains of different maize varieties.

The study of gene expression in maize varieties represents a powerful tool aiming to increase vitamin A precursors. However, the isolation of RNA from different maize varieties is challenging because these varieties show different levels of polysaccharides, and most methods available for RNA isolation are inappropriate for grain samples. The polysaccharides co-purify and co-precipitate with RNA during isolation, resulting in low-quality RNA, compromising the use of RNA in subsequent applications. Thus, a cetyltrimethylammonium bromide (CTAB)-based method was adapted in this study and compared with six methods for RNA isolation, including commercial reagents and RNA and DNA isolation kits, in order to identify the most appropriate for maize grains from different varieties. Most of the methods evaluated were considered inadequate due to limitations in terms of purity and/or quantity of the isolated RNA, which affected the efficiency of subsequent RT-qPCR analysis, resulting in nonamplification of ß-carotene hydroxylase gene (HYD3) or high deviation among replicates. However, the CTAB modified method allowed the study to obtain intact RNA, with high quality and quantity, from 25 maize varieties. Furthermore, this RNA was successfully used to evaluate the expression of HYD3 gene by real-time qualitative polymerase chain reaction (RT-qPCR), and thus represents a simple, efficient, and low-cost strategy.

Carotenoid biosynthetic and catabolic pathways: gene expression and carotenoid content in grains of maize landraces.

Plant carotenoids have been implicated in preventing several age-related diseases, and they also provide vitamin A precursors; therefore, increasing the content of carotenoids in maize grains is of great interest. It is not well understood, however, how the carotenoid biosynthetic pathway is regulated. Fortunately, the maize germplasm exhibits a high degree of genetic diversity that can be exploited for this purpose. Here, the accumulation of carotenoids and the expression of genes from carotenoid metabolic and catabolic pathways were investigated in several maize landraces. The carotenoid content in grains varied from 10.03, in the white variety MC5, to 61.50 µg·g⁻¹, in the yellow-to-orange variety MC3, and the major carotenoids detected were lutein and zeaxanthin. PSY1 (phythoene synthase) expression showed a positive correlation with the total carotenoid content. Additionally, the PSY1 and HYD3 (ferredoxin-dependent di-iron monooxygenase) expression levels were positively correlated with ß-cryptoxanthin and zeaxanthin, while CYP97C (cytochrome P450-type monooxygenase) expression did not correlate with any of the carotenoids. In contrast, ZmCCD1 (carotenoid dioxygenase) was more highly expressed at the beginning of grain development, as well as in the white variety, and its expression was inversely correlated with the accumulation of several carotenoids, suggesting that CCD1 is also an important enzyme to be considered when attempting to improve the carotenoid content in maize. The MC27 and MC1 varieties showed the highest HYD3/CYP97C ratios, suggesting that they are promising candidates for increasing the zeaxanthin content; in contrast, MC14 and MC7 showed low HYD3/CYP97C, suggesting that they may be useful in biofortification efforts aimed at promoting the accumulation of provitamin A. The results of this study demonstrate the use of maize germplasm to provide insight into the regulation of genes involved in the carotenoid pathway, which would thus better enable us to select promising varieties for biofortification efforts.

Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains.

KEY MESSAGE: The stability of candidate reference genes was evaluated in maize landrace varieties and during multiple grain developmental stages to evaluate the expression of carotenoid-related genes by RT-qPCR for application to maize biofortification. Vitamin A deficiency affects millions of children worldwide; therefore, increasing the content of vitamin A precursors in maize grains is of interest. The study of the expression of genes involved in the carotenoid biosynthetic pathway in maize grains has provided useful information for metabolic engineering approaches. However, reliable results using real-time quantitative polymerase chain reaction (RT-qPCR) experiments are dependent on the use of the appropriate reference genes. In this study, we utilized geNorm and NormFinder softwares to identify the most stably expressed candidate reference genes in samples from seven stages of grain development and from eight landrace varieties. The results of the analysis performed using geNorm indicated that tubulin (TUB) and actin (ACT) were the most suitable reference genes among all experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) showed the least stability. The same result was obtained with the NormFinder software. The minimum number of genes required in each experimental condition to normalize the gene expression data was also determined by geNorm. The expression of phytoene synthase gene (PSY1), the first enzyme in the carotenoid biosynthetic pathway, was overestimated when the least stable candidate gene (GAPDH) was used as the internal control instead of the most stable gene pair (ACT + TUB), thus highlighting the importance of validating reference genes before conducting a RT-qPCR experiment to obtain accurate results. This study is the first survey of the stability of genes for use as reference genes to normalize RT-qPCR data from maize landraces during multiple stages of grain development.

Larval chondrocranium morphology of five species of Proceratophrys Miranda-Ribeiro (Amphibia; Anura; Odontophrynidae).

The chondrocranium and hyobranchial morphology are described for the tadpoles of five species of Proceratophrys Miranda-Ribeiro. The chondrocranium of P. appendiculata, P boiei, P laticeps and P. tupinamba are very similar and can be distinguished from that of P. cristiceps by: 1) less developed processus muscularis; 2) thinner palatoquadrate; 3) broadest ethmoidal region; 4) longer cornua trabeculae; and 5) morphology f the suprarostral cartilages. A morphological variation among Proceratophrys boiei is described. It reveals the existence of at least three distinct evolutionary lineages under this name. A brief comparison with other cycloramphid species is also provided.