Nezara viridula (Hemiptera: Pentatomidae) transcriptomic analysis and neuropeptidomics

Stinkbugs (Hemiptera: Pentatomidae) are of major economic importance as pest of crops. Among the species composing the stinkbug complex, Nezara viridula is one of the most abundant in Brazil, Argentina and the Southern USA. However, this species has been poorly characterized at the genetic and physiological level. Here we sequenced and analyzed the complete transcriptome of N. viridula male and female adults. We identified neuropeptide precursor genes and G-protein coupled receptors for neuropeptides in this transcriptome. Mature neuropeptides were identified in N. viridula brain extracts by liquid chromatography-tandem mass spectrometry. We also analyzed the neuropeptide precursor complement in the genome sequence of Halyomorpha halys, another pentatomid of economic relevance. We compared the results in both pentatomids with the well-characterized neuropeptide repertoire from the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae). We identified both group-specific features (which could be related to the different feeding habits) and similarities that could be characteristic of Heteroptera. This work contributes to a deeper knowledge of the genetic information of these pests, with a focus on neuroendocrine system characterization.Fil: Lavore, Andres Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Pérez Gianmarco, Lucila Maité. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Esponda Behrens, Natalia Irene. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Palacio, Victorio Gabriel. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Catalano, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Rivera Pomar, Rolando. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; ArgentinaFil: Ons, Sheila. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Adaptations in energy metabolism and gene family expansions revealed by comparative transcriptomics of three Chagas disease triatomine vectors

Background: Chagas disease is a parasitic infection caused by Trypanosoma cruzi. It is an important public health problem affecting around seven to eight million people in the Americas. A large number of hematophagous triatomine insect species, occupying diverse natural and human-modified ecological niches transmit this disease. Triatomines are long-living hemipterans that have evolved to explode different habitats to associate with their vertebrate hosts. Understanding the molecular basis of the extreme physiological conditions including starvation tolerance and longevity could provide insights for developing novel control strategies. We describe the normalized cDNA, full body transcriptome analysis of three main vectors in North, Central and South America, Triatoma pallidipennis, T. dimidiata and T. infestans. Results: Two-thirds of the de novo assembled transcriptomes map to the Rhodnius prolixus genome and proteome. A Triatoma expansion of the calycin family and two types of protease inhibitors, pacifastins and cystatins were identified. A high number of transcriptionally active class I transposable elements was documented in T. infestans, compared with T. dimidiata and T. pallidipennis. Sequence identity in Triatoma-R. prolixus 1:1 orthologs revealed high sequence divergence in four enzymes participating in gluconeogenesis, glycogen synthesis and the pentose phosphate pathway, indicating high evolutionary rates of these genes. Also, molecular evidence suggesting positive selection was found for several genes of the oxidative phosphorylation I, III and V complexes. Conclusions: Protease inhibitors and calycin-coding gene expansions provide insights into rapidly evolving processes of protease regulation and haematophagy. Higher evolutionary rates in enzymes that exert metabolic flux control towards anabolism and evidence for positive selection in oxidative phosphorylation complexes might represent genetic adaptations, possibly related to prolonged starvation, oxidative stress tolerance, longevity, and hematophagy and flight reduction. Overall, this work generated novel hypothesis related to biological adaptations to extreme physiological conditions and diverse ecological niches that sustain Chagas disease transmission.Fil: Martínez Barnetche, Jesús. Instituto Nacional de Salud Pública; MéxicoFil: Lavore, Andres Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Beliera, Melina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Téllez Sosa, Juan. Instituto Nacional de Salud Pública; MéxicoFil: Zumaya Estrada, Federico A.. Instituto Nacional de Salud Pública; MéxicoFil: Palacio, Victorio Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Godoy Lozano, Ernestina. Instituto Nacional de Salud Pública; MéxicoFil: Rivera Pomar, Rolando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Rodríguez, Mario Henry. Instituto Nacional de Salud Pública; Méxic

Secuenciación y análisis del transcriptoma de dalbulusmaidis

Los auquenorrincos (chicharritas o cotorritas) son insectos exclusivamente fitófagos, que pueden causar importantes daños económicos sobre los cultivos. Una de las enfermedades vectorizadas por ellos es el achaparramiento del maíz o Corn Stunt Disease, potencialmente una de las enfermedades más serias del cultivo de maíz, capaz de causar pérdidas parciales o totales en la producci ón en las zonas afectadas. En Argentina, Dalbulusmaidis (Hemiptera: Auchenorrhyncha) es el único vector a campo conocido como transmisor del Spiroplasmakunkelii , patógeno causal del Corn Stunt . Dada su importancia como plaga en la agricultura, se secuenció el transcriptoma de todos los estadios del ciclo de vida de este insecto (huevos, 5 estadios ninfales y dos muestras de adultos). Se utilizó un pool de insectos para abarcar la mayor cantidad de genes expresados. Como la información genómica de Dalbulusma idis no está disponible, se realizó el ensamblado de novo . Se compararon los ensambles realizados con 3 programas: VELVET OASES, ABySS y Trinity. Se evaluaron utilizando métricas (N50, longitud de contig ) y medidas de cobertura (CEG, BUSCO). En base a es tosanálisis, se decidió buscar genes del desarrollo en los ensambles de VELVET OASES y Trinity. El porcentaje total de genes encontrado fue mayor para el ensamble de Trinity. Teniendo en cuenta los resultados previos, se ensamblaron el resto de las muest ras con Trinity, obteniendo valores de métricas y coberturas muy buenos. Además se compararon los transcriptomas con proteomas publicados como medida de homología entre especies. En este trabajo se compararon distintos métodos de ensamble de novo y se selec cionó el que mejor se adaptó a nuestros datos y experimentosFil: Palacio, Victorio Gabriel. Universidad Nacional del Noroeste de la Provincia de Buenos AiresFil: Lavore, Andrés. Universidad Nacional del Noroeste de la Provincia de Buenos AiresFil: Catalano, María Inés . Universidad Nacional del Noroeste de la Provincia de Buenos AiresFil: Rivera Pomar, Rolando . Universidad Nacional del Noroeste de la Provincia de Buenos Aire

Conditions and Environment of Work in Two Recycling Centers in the Metropolitan Area of Buenos Aires

INTRODUCCIÓN: Las plantas de clasificación de residuos aparecen como una alternativa de inserción para los cartoneros. No obstante, se advierten falencias que producen efectos perjudiciales sobre la salud y el ambiente laboral de estos trabajadores no clásicos. OBJETIVOS: Describir el proceso, las condiciones y el medio ambiente de trabajo en centros de acopio y separación de reciclables en el Área Metropolitana de Buenos Aires (AMBA), evaluando las exigencias físicas y el riesgo sanitario. MÉTODOS: Se realizó un estudio descriptivo en dos establecimientos del AMBA, uno en contexto urbano y el otro en descampado periurbano. Se efectuó trabajo de campo etnográfico con observación, entrevistas y dinámicas grupales con técnicas proyectivas y eutonía, en paralelo con muestreos biológicos. RESULTADOS: Se identificaron situaciones externas de riesgo y otras propias del trabajador definidas como exigencias. No se encontraron virus, aunque sí vectores de dengue y encefalitis San Luis. Existe circulación de parásitos con reservorio en roedores, perros y gatos. Se documentaron otros riesgos asociados a residuos industriales, deficiente provisión de agua e incendio. Se registró sobrecarga sobre los trabajadores varones. CONCLUSIONES: Se sugiere vigilancia sanitaria en perros, gatos, roedores e insectos, así como muestreo aleatorio de materiales ingresados. Para reducir exigencias se indica rotación entre puestos y uso de máquinas simples.INTRODUCTION: Recycling centers appear as an alternative for the employability of informal garbage collectors. However, there are deficiencies producing harmful effects on human health and the labor environment of these non-conventional workers. OBJECTIVES: To describe the process, conditions and environment of work of recycling centers in the Metropolitan Area of Buenos Aires (MABA), evaluating physical demands and health risk. METHODS: A descriptive study was conducted in two recycling facilities located in the MABA, one in an urban setting and the other one in a peri-urban area. Ethnographic fieldwork was carried out with observation, interviews and group dynamics with projective techniques and eutony, in parallel with biological sampling. RESULTS: External risk situations were identified, along with other worker-related factors defined as demands. No viruses were found, although there are vectors of dengue and Saint Louis encephalitis. There is also circulation of parasites with reservoir in rodents, dogs and cats. Other risks associated with industrial waste, poor water supply and fire were documented. There was an overload on male workers. CONCLUSIONS: Sanitary surveillance is suggested in dogs, cats, rodents and insects, as well as random sampling of incoming materials. Job rotation and use of simple machines are indicated to reduce demands.Centro de Estudios Parasitológicos y de Vectore

Conditions and Environment of Work in Two Recycling Centers in the Metropolitan Area of Buenos Aires

INTRODUCCIÓN: Las plantas de clasificación deresiduos aparecen como una alternativa de inserción para loscartoneros. No obstante, se advierten falencias que producenefectos perjudiciales sobre la salud y el ambiente laboral deestos trabajadores no clásicos. OBJETIVOS: Describir el proceso,las condiciones y el medio ambiente de trabajo en centros deacopio y separación de reciclables en el Área Metropolitana deBuenos Aires (AMBA), evaluando las exigencias físicas y el riesgosanitario. MÉTODOS: Se realizó un estudio descriptivo en dosestablecimientos del AMBA, uno en contexto urbano y el otro endescampado periurbano. Se efectuó trabajo de campo etnográficocon observación, entrevistas y dinámicas grupales con técnicasproyectivas y eutonía, en paralelo con muestreos biológicos.RESULTADOS: Se identificaron situaciones externas de riesgo y otraspropias del trabajador definidas como exigencias. No se encontraronvirus, aunque sí vectores de dengue y encefalitis San Luis. Existecirculación de parásitos con reservorio en roedores, perros y gatos.Se documentaron otros riesgos asociados a residuos industriales,deficiente provisión de agua e incendio. Se registró sobrecargasobre los trabajadores varones. CONCLUSIONES: Se sugierevigilancia sanitaria en perros, gatos, roedores e insectos, así comomuestreo aleatorio de materiales ingresados. Para reducir exigenciasse indica rotación entre puestos y uso de máquinas simples.Fil: Mastrangelo, Andrea Veronica. Universidad Nacional de San Martín. Instituto de Altos Estudios Sociales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Schamber, Pablo Javier. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbran". Instituto Nacional de Enfermedades Virales Humanas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lizuain, Arturo Andrés. Dirección Nacional de Instituto de Investigación; ArgentinaFil: Guerreiro Martins, Natalia Beatriz. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; ArgentinaFil: Palacio, Victorio Gabriel. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); Argentin

Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity.

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs

Comparative and functional triatomine genomics reveals reductions and expansions in insecticide resistance-related gene families

Background: Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas’ disease. This is a neglected disease affecting approximately 8 million people in Latin America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas’ disease. Methods and findings: The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas’ disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic detoxification mechanisms on the high levels of pyrethroid resistance detected in triatomines from the Gran Chaco ecoregion. Conclusions and significance: Our results help to elucidate the potential insecticide resistance mechanisms in vectors of Chagas’ disease and provide new relevant information for this field. This study shows that metabolic resistance might be a contributing cause of the high pyrethroid resistance observed in wild T. infestans populations from the Gran Chaco ecoregion, area in which although subjected to intense pyrethroid treatments, vector control has failed. This study opens new avenues for further functional studies on triatomine detoxification mechanisms.Centro Regional de Estudios GenómicosCentro de Endocrinología Experimental y Aplicad

Comparative and functional triatomine genomics reveals reductions and expansions in insecticide resistance-related gene families

Background: Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas’ disease. This is a neglected disease affecting approximately 8 million people in Latin America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas’ disease. Methods and findings: The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas’ disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic detoxification mechanisms on the high levels of pyrethroid resistance detected in triatomines from the Gran Chaco ecoregion. Conclusions and significance: Our results help to elucidate the potential insecticide resistance mechanisms in vectors of Chagas’ disease and provide new relevant information for this field. This study shows that metabolic resistance might be a contributing cause of the high pyrethroid resistance observed in wild T. infestans populations from the Gran Chaco ecoregion, area in which although subjected to intense pyrethroid treatments, vector control has failed. This study opens new avenues for further functional studies on triatomine detoxification mechanisms.Centro Regional de Estudios GenómicosCentro de Endocrinología Experimental y Aplicad

Adaptations in energy metabolism and gene family expansions revealed by comparative transcriptomics of three Chagas disease triatomine vectors

Background: Chagas disease is a parasitic infection caused by Trypanosoma cruzi. It is an important public health problem affecting around seven to eight million people in the Americas. A large number of hematophagous triatomine insect species, occupying diverse natural and human-modified ecological niches transmit this disease. Triatomines are long-living hemipterans that have evolved to explode different habitats to associate with their vertebrate hosts. Understanding the molecular basis of the extreme physiological conditions including starvation tolerance and longevity could provide insights for developing novel control strategies. We describe the normalized cDNA, full body transcriptome analysis of three main vectors in North, Central and South America, Triatoma pallidipennis, T. dimidiata and T. infestans. Results: Two-thirds of the de novo assembled transcriptomes map to the Rhodnius prolixus genome and proteome. A Triatoma expansion of the calycin family and two types of protease inhibitors, pacifastins and cystatins were identified. A high number of transcriptionally active class I transposable elements was documented in T. infestans, compared with T. dimidiata and T. pallidipennis. Sequence identity in Triatoma-R. prolixus 1:1 orthologs revealed high sequence divergence in four enzymes participating in gluconeogenesis, glycogen synthesis and the pentose phosphate pathway, indicating high evolutionary rates of these genes. Also, molecular evidence suggesting positive selection was found for several genes of the oxidative phosphorylation I, III and V complexes. Conclusions: Protease inhibitors and calycin-coding gene expansions provide insights into rapidly evolving processes of protease regulation and haematophagy. Higher evolutionary rates in enzymes that exert metabolic flux control towards anabolism and evidence for positive selection in oxidative phosphorylation complexes might represent genetic adaptations, possibly related to prolonged starvation, oxidative stress tolerance, longevity, and hematophagy and flight reduction. Overall, this work generated novel hypothesis related to biological adaptations to extreme physiological conditions and diverse ecological niches that sustain Chagas disease transmission.Centro Regional de Estudios Genómico

Comparative and functional triatomine genomics reveals reductions and expansions in insecticide resistance-related gene families

Background: Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas’ disease. This is a neglected disease affecting approximately 8 million people in Latin America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas’ disease. Methods and findings: The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas’ disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic detoxification mechanisms on the high levels of pyrethroid resistance detected in triatomines from the Gran Chaco ecoregion. Conclusions and significance: Our results help to elucidate the potential insecticide resistance mechanisms in vectors of Chagas’ disease and provide new relevant information for this field. This study shows that metabolic resistance might be a contributing cause of the high pyrethroid resistance observed in wild T. infestans populations from the Gran Chaco ecoregion, area in which although subjected to intense pyrethroid treatments, vector control has failed. This study opens new avenues for further functional studies on triatomine detoxification mechanisms.Centro Regional de Estudios GenómicosCentro de Endocrinología Experimental y Aplicad