Biobanking and use of gonadal tissues - a promising strategy for conserving wildlife from the Caatinga biome

Biological Resource Banks (BRB) or Genetic Resource Banks (GRB) are critical tools for the conservation of animal biodiversity. According to the International Union for Conservation of Nature, more than 38,500 species are threatened with extinction, out of a total of 138,300 surveyed species. These banks are repositories of biological samples and data recovered and preserved for the long term by zoos, universities, research centers and other conservation organizations. In recent years, BRB have increasingly included ovarian and testicular tissues as additional options to rescue and propagate wild species, especially those at risk of extinction. After in vitro culture or grafting, gonadal tissues are potential sources of matured gametes that can be used for Assisted Reproduction Technologies while informing about gametogenesis or mechanisms involved in infertility. It therefore is crucial to properly recover, cryopreserve, and culture these tissues using species-specific protocols. Developing BRBs is currently one of the strategies to preserve species from the Caatinga biome - an exclusively Brazilian biome with a rich wild fauna that suffers from anthropogenic activities. Among wild species from this biome, studies have been primarily conducted in collared peccaries, agoutis, cavies, and armadillos to preserve their ovarian and testicular tissues. Additionally, domestic species such as the domestic cat and donkeys have been proposed as models for wild species that are phylogenetically close. This review addresses the main technical aspects involved in obtaining BRB derived from gonadal tissues in some wild species of the Caatinga biome. It reports recent advances and perspectives to use these biological materials for wildlife conservation.(AU)

Conservation, animal behaviour, and human-animal relationship in zoos. Why is animal welfare so important?

Although zoos are committed to wildlife conservation and have a long-term positive impact on visitors' attitudes towards wildlife, the question of whether maintaining wild animals in human care is justified remains as animal welfare concerns grow and human understanding of animal intelligence and capacities broadens. Zoos have always been the subject of debate, with conflicts between those who argue they save endangered species and educate visitors, and animal rights activists who believe that conditions of wild animals are inadequate and that zoos should not exist. In this review, we do not discuss the moral side of the issue, but the scientific one. This manuscript aims to show the scope of literature available on the strengths and weaknesses of modern zoos regarding wild animal welfare. We provide information useful to argue why zoos are important in modern society and factors that influence welfare are examined. Some potentially stressful stimuli may diminish animal welfare in zoo animals, while some of the benefits zoos offer to conservation and science include the opportunity to study and learn about different aspects necessary to improve management practices; the possibility of breeding wild animals in zoos has been a key factor in the recovery of species that have improved their conservation status. Animal welfare is an essential part of wildlife conservation, so efforts should be directed to ensure the best possible quality of life and optimum conditions of all zoo animals in our care.(AU)

Conservation, animal behaviour, and human-animal relationship in zoos. Why is animal welfare so important?

Although zoos are committed to wildlife conservation and have a long-term positive impact on visitors' attitudes towards wildlife, the question of whether maintaining wild animals in human care is justified remains as animal welfare concerns grow and human understanding of animal intelligence and capacities broadens. Zoos have always been the subject of debate, with conflicts between those who argue they save endangered species and educate visitors, and animal rights activists who believe that conditions of wild animals are inadequate and that zoos should not exist. In this review, we do not discuss the moral side of the issue, but the scientific one. This manuscript aims to show the scope of literature available on the strengths and weaknesses of modern zoos regarding wild animal welfare. We provide information useful to argue why zoos are important in modern society and factors that influence welfare are examined. Some potentially stressful stimuli may diminish animal welfare in zoo animals, while some of the benefits zoos offer to conservation and science include the opportunity to study and learn about different aspects necessary to improve management practices; the possibility of breeding wild animals in zoos has been a key factor in the recovery of species that have improved their conservation status. Animal welfare is an essential part of wildlife conservation, so efforts should be directed to ensure the best possible quality of life and optimum conditions of all zoo animals in our care.(AU)

Conservation, animal behaviour, and human-animal relationship in zoos. Why is animal welfare so important?

Although zoos are committed to wildlife conservation and have a long-term positive impact on visitors' attitudes towards wildlife, the question of whether maintaining wild animals in human care is justified remains as animal welfare concerns grow and human understanding of animal intelligence and capacities broadens. Zoos have always been the subject of debate, with conflicts between those who argue they save endangered species and educate visitors, and animal rights activists who believe that conditions of wild animals are inadequate and that zoos should not exist. In this review, we do not discuss the moral side of the issue, but the scientific one. This manuscript aims to show the scope of literature available on the strengths and weaknesses of modern zoos regarding wild animal welfare. We provide information useful to argue why zoos are important in modern society and factors that influence welfare are examined. Some potentially stressful stimuli may diminish animal welfare in zoo animals, while some of the benefits zoos offer to conservation and science include the opportunity to study and learn about different aspects necessary to improve management practices; the possibility of breeding wild animals in zoos has been a key factor in the recovery of species that have improved their conservation status. Animal welfare is an essential part of wildlife conservation, so efforts should be directed to ensure the best possible quality of life and optimum conditions of all zoo animals in our care.

Survey of Toxoplasma gondii, Neospora caninum and Sarcocystis neurona antibodies in wild red-tailed Amazon parrots (Amazona brasiliensis) / Pesquisa de anticorpos anti-Toxoplasma gondii, anti-Neospora caninum e anti-Sarcocystis neurona em papagaios-de-cara-roxa (Amazona brasiliensis) de vida livre

Toxoplasma gondii, Neospora caninum and Sarcocystis neurona are obligate intracellular parasites within the phylum Apicomplexa. The red-tailed Amazon parrot (Amazona brasiliensis) is a near-threatened species of psittacine that is endemic to the Atlantic Forest of Brazil and has been designated as a bioindicator because of its sensitivity to environmental qualitative status and changes. The aim of this study was to evaluate the presence of antibodies against T. gondii, N. caninum and S. neurona in wild red-tailed Amazon parrot nestlings on Rasa Island, Brazil. Blood samples were collected from 51 parrots and plasma samples were stored at 20 °C until immunofluorescence antibody tests (IFAT) were performed. Antigen slides were prepared using tachyzoites of T. gondii (RH strain) and, N. caninum (NC-1 strain) and using merozoites of S. neurona (SNR37 strain). Plasma samples were tested at initial dilutions of 1:16 for T. gondii, 1:50 for N. caninum and 1:5 for S. neurona. An anti-chicken antibody conjugated with FITC was used as a secondary antibody at 1:50 dilution. No antibodies for any of these three protozoa were found, thus suggesting that these wild red-tailed Amazon parrot nestlings had not been exposed to these parasites.(AU)

Toxoplasma gondii, Neospora caninum e Sarcocystis neurona são protozoários intracelulares do filo Apicomplexa. O papagaio-de-cara-roxa (Amazona brasiliensis) é um psitacídeo endêmico da floresta atlântica, considerado uma espécie quase ameaçada de extinção e bioindicadora por sua sensibilidade às mudanças no ambiente. O objetivo do presente estudo foi detectar a presença de anticorpos contra T. gondii, N. caninum e S. neurona em filhotes de papagaios-de-cara-roxa (Amazona brasiliensis) de vida livre na Ilha Rasa, Brasil. Amostras de sangue foram coletadas de 51 papagaios e armazenadas a - 20ºC até a realização da Reação de Imunofluorescência Indireta (RIFI). As lâminas de RIFI com os antígenos, foram preparadas com taquizoítos de T. gondii (cepa RH) e N. caninum (cepa NC-1) e com merozoítos de S. neurona (cepa SNR37). Os plasmas foram diluídos em PBS (Ph 7,2) nas diluições 1:16 para T. gondii, 1:50 para N. caninum e 1:5 para S. neurona. O conjugado anti-IgG de galinhas marcado com fluoresceína (FITC) foi utilizado na diluição de 1:50. Não foram detectados anticorpos para os três protozoários nas amostras sugerindo que os filhotes de papagaios-de-cara-roxa não foram expostos aos protozoários.(AU)

Causes of mortality of seabirds stranded at the Northeastern coast of Brazil / Causas de mortalidade de aves marinhas encalhadas na costa do Nordeste do Brasil

The aim of this work was to determine the main species of stranded seabirds at the Northeastern coast of Brazil in addition to the most frequent causes of stranding and mortality. The study was conducted in a monitored area for three years (2012-2014), from the coastline of south Alagoas through north coast of Bahia encompassing 254km of coast. The seabirds found alive during the monitoring were sent to rehabilitation, clinically examined and the carcasses were removed, necropsied and histopathologically analyzed. A total of 1.347 seabirds were found stranded. Of these, 378 were found alive and sent to rehabilitation. From the 969 dead seabirds 806 were unsuitable for necropsy, being only 163 submitted to necropsy and histopathological analysis. Calonectris borealis, Puffinus gravis and Puffinus puffinus were the main seabirds stranded in the studied area. Most stranding occurred from March to June with an increase during April and May for the most species of seabirds. The main clinical signs of stranded seabirds consisted of inappetence, apathy, low body score, hypothermia, flying or movement difficulty and prolonged recumbency. Natural causes followed by infectious diseases and anthropogenic environmental factors were the main causes of death of seabirds stranded on the Northeastern coast of Brazil.(AU)

O objetivo deste trabalho foi determinar as principais espécies de aves marinhas encalhadas na costa do Nordeste do Brasil, bem como as mais frequentes causas de encalhe e mortalidade. O estudo foi desenvolvido em área monitorada durante três anos (2012-2014), entre o litoral sul de Alagoas e o litoral norte da Bahia, perfazendo um total de 254km de extensão da costa. As aves encontradas vivas foram encaminhadas para reabilitação e examinadas clinicamente; as carcaças foram removidas, necropsiadas e os órgãos foram analisados por meio da histopatologia. Um total de 1.347 aves foi encontrado, encalhadas. Dessas, 378 estavam vivas e foram encaminhadas para a reabilitação. Das 969 aves marinhas mortas, 806 estavam impróprias para realização de necropsia, sendo apenas 163 submetidas à necropsia e análise histopatológica. Calonectris borealis, Puffinus gravis e Puffinus puffinus foram as principais espécies encontradas na área estudada. A maioria dos encalhes para grande parte das aves marinhas ocorreram de março a junho, com um aumento durante abril e maio. Os principais sinais clínicos das aves marinhas consistiram em inapetência, apatia, baixo escore corporal, hipotermia, dificuldade de voo ou movimento e decúbito prolongado. Causas naturais, seguidas de doenças infecciosas e fatores antropogênicos, foram as principais causas de mortes de aves marinhas encalhadas na costa do Nordeste do Brasil.(AU)

Causes of mortality of seabirds stranded at the Northeastern coast of Brazil / Causas de mortalidade de aves marinhas encalhadas na costa do Nordeste do Brasil

The aim of this work was to determine the main species of stranded seabirds at the Northeastern coast of Brazil in addition to the most frequent causes of stranding and mortality. The study was conducted in a monitored area for three years (2012-2014), from the coastline of south Alagoas through north coast of Bahia encompassing 254km of coast. The seabirds found alive during the monitoring were sent to rehabilitation, clinically examined and the carcasses were removed, necropsied and histopathologically analyzed. A total of 1.347 seabirds were found stranded. Of these, 378 were found alive and sent to rehabilitation. From the 969 dead seabirds 806 were unsuitable for necropsy, being only 163 submitted to necropsy and histopathological analysis. Calonectris borealis, Puffinus gravis and Puffinus puffinus were the main seabirds stranded in the studied area. Most stranding occurred from March to June with an increase during April and May for the most species of seabirds. The main clinical signs of stranded seabirds consisted of inappetence, apathy, low body score, hypothermia, flying or movement difficulty and prolonged recumbency. Natural causes followed by infectious diseases and anthropogenic environmental factors were the main causes of death of seabirds stranded on the Northeastern coast of Brazil.(AU)

O objetivo deste trabalho foi determinar as principais espécies de aves marinhas encalhadas na costa do Nordeste do Brasil, bem como as mais frequentes causas de encalhe e mortalidade. O estudo foi desenvolvido em área monitorada durante três anos (2012-2014), entre o litoral sul de Alagoas e o litoral norte da Bahia, perfazendo um total de 254km de extensão da costa. As aves encontradas vivas foram encaminhadas para reabilitação e examinadas clinicamente; as carcaças foram removidas, necropsiadas e os órgãos foram analisados por meio da histopatologia. Um total de 1.347 aves foi encontrado, encalhadas. Dessas, 378 estavam vivas e foram encaminhadas para a reabilitação. Das 969 aves marinhas mortas, 806 estavam impróprias para realização de necropsia, sendo apenas 163 submetidas à necropsia e análise histopatológica. Calonectris borealis, Puffinus gravis e Puffinus puffinus foram as principais espécies encontradas na área estudada. A maioria dos encalhes para grande parte das aves marinhas ocorreram de março a junho, com um aumento durante abril e maio. Os principais sinais clínicos das aves marinhas consistiram em inapetência, apatia, baixo escore corporal, hipotermia, dificuldade de voo ou movimento e decúbito prolongado. Causas naturais, seguidas de doenças infecciosas e fatores antropogênicos, foram as principais causas de mortes de aves marinhas encalhadas na costa do Nordeste do Brasil.(AU)

Causes of mortality of seabirds stranded at the Northeastern coast of Brazil

ABSTRACT: The aim of this work was to determine the main species of stranded seabirds at the Northeastern coast of Brazil in addition to the most frequent causes of stranding and mortality. The study was conducted in a monitored area for three years (2012-2014), from the coastline of south Alagoas through north coast of Bahia encompassing 254km of coast. The seabirds found alive during the monitoring were sent to rehabilitation, clinically examined and the carcasses were removed, necropsied and histopathologically analyzed. A total of 1.347 seabirds were found stranded. Of these, 378 were found alive and sent to rehabilitation. From the 969 dead seabirds 806 were unsuitable for necropsy, being only 163 submitted to necropsy and histopathological analysis. Calonectris borealis, Puffinus gravis and Puffinus puffinus were the main seabirds stranded in the studied area. Most stranding occurred from March to June with an increase during April and May for the most species of seabirds. The main clinical signs of stranded seabirds consisted of inappetence, apathy, low body score, hypothermia, flying or movement difficulty and prolonged recumbency. Natural causes followed by infectious diseases and anthropogenic environmental factors were the main causes of death of seabirds stranded on the Northeastern coast of Brazil.

RESUMO: O objetivo deste trabalho foi determinar as principais espécies de aves marinhas encalhadas na costa do Nordeste do Brasil, bem como as mais frequentes causas de encalhe e mortalidade. O estudo foi desenvolvido em área monitorada durante três anos (2012-2014), entre o litoral sul de Alagoas e o litoral norte da Bahia, perfazendo um total de 254km de extensão da costa. As aves encontradas vivas foram encaminhadas para reabilitação e examinadas clinicamente; as carcaças foram removidas, necropsiadas e os órgãos foram analisados por meio da histopatologia. Um total de 1.347 aves foi encontrado, encalhadas. Dessas, 378 estavam vivas e foram encaminhadas para a reabilitação. Das 969 aves marinhas mortas, 806 estavam impróprias para realização de necropsia, sendo apenas 163 submetidas à necropsia e análise histopatológica. Calonectris borealis, Puffinus gravis e Puffinus puffinus foram as principais espécies encontradas na área estudada. A maioria dos encalhes para grande parte das aves marinhas ocorreram de março a junho, com um aumento durante abril e maio. Os principais sinais clínicos das aves marinhas consistiram em inapetência, apatia, baixo escore corporal, hipotermia, dificuldade de voo ou movimento e decúbito prolongado. Causas naturais, seguidas de doenças infecciosas e fatores antropogênicos, foram as principais causas de mortes de aves marinhas encalhadas na costa do Nordeste do Brasil.

Endoparasites in domestic animals surrounding an Atlantic Forest remnant, in São Paulo State, Brazil

Morro do Diabo State Park (MDSP) is a significant remnant of the Atlantic Rain Forest in Brazil and is surrounded by rural properties. In that area, wild and domestic animals and humans are in close contact, which facilitates the two-way flow of infectious diseases among them. We assessed endoparasites in domestic livestock from all rural properties surrounding MDSP. There were sampled 197 cattle, 37 horses, 11 sheep, 25 swine, 21 dogs, one cat and 62 groups of chickens from 10 large private properties and 75 rural settlements. Eimeria spp. was present in almost all hosts, excepted in horses, dogs and cats. Giardia cysts were present only in cattle. Nematodes were found in swine, ruminants and horses in high prevalence. Ancylostoma, Toxocara and Sarcocystis were found in dogs. Chickens were found with coccidia, Ascaridida and Capillaria spp.. These parasites can cause malnutrition and reproductive disorders for their hosts. Strategies to prevent and control the spread of endoparasites can improve wildlife, animal and human health in this area.(AU)

O Parque Estadual Morro do diabo (PEMD) é um significante remanescente de Mata Atlântica no Brasil, e rodeado de propriedades rurais. Nesta área humanos, animais domésticos e silvestres vivem próximos, o que facilita o fluxo de agentes infecciosas entre eles. Nós avaliamos a presença de endoparasitas, por meio de exame coproparasitológico dos animais domésticos de todas as propriedades rurais do entorno do PEMD. Foram amostrados 197 bovinos, 37 equinos, 11 ovinos, 25 suínos, 62 grupos de galinhas, 22 cães e 1 gato, residentes em 10 grandes propriedades privadas e 75 assentamentos rurais. Eimeria spp. estava presente em quase todas as espécies hospedeiras, com excessão de equinos, cães e gatos. Cistos de Giardia estavam presentes somente em bovinos. Nematodes foram encontrados em suínos, ruminantes e equinos em alta prevalência. Ancylostoma, Toxocara e Sarcocystis foram encontrados em cães. Galinhas foram encontradas com coccidia, Ascaridida e Capillaria spp.. Os parasitas encontrados podem causar má nutrição e problemas reprodutivos para seus hospedeiros. Medidas de prevenção e controle da dispersão de endoparasitas podem melhorar a condição de saúde pública, animal e ambiental nesta área.(AU)

Endoparasites in domestic animals surrounding an Atlantic Forest remnant, in São Paulo State, Brazil / Endoparasitas em animais domésticos que vivem ao redor de uma reserva florestal, no Estado de São Paulo, Brasil

Morro do Diabo State Park (MDSP) is a significant remnant of the Atlantic Rain Forest in Brazil and is surrounded by rural properties. In that area, wild and domestic animals and humans are in close contact, which facilitates the two-way flow of infectious diseases among them. We assessed endoparasites in domestic livestock from all rural properties surrounding MDSP. There were sampled 197 cattle, 37 horses, 11 sheep, 25 swine, 21 dogs, one cat and 62 groups of chickens from 10 large private properties and 75 rural settlements. Eimeria spp. was present in almost all hosts, excepted in horses, dogs and cats. Giardia cysts were present only in cattle. Nematodes were found in swine, ruminants and horses in high prevalence. Ancylostoma, Toxocara and Sarcocystis were found in dogs. Chickens were found with coccidia, Ascaridida and Capillaria spp.. These parasites can cause malnutrition and reproductive disorders for their hosts. Strategies to prevent and control the spread of endoparasites can improve wildlife, animal and human health in this area.(AU)

O Parque Estadual Morro do diabo (PEMD) é um significante remanescente de Mata Atlântica no Brasil, e rodeado de propriedades rurais. Nesta área humanos, animais domésticos e silvestres vivem próximos, o que facilita o fluxo de agentes infecciosas entre eles. Nós avaliamos a presença de endoparasitas, por meio de exame coproparasitológico dos animais domésticos de todas as propriedades rurais do entorno do PEMD. Foram amostrados 197 bovinos, 37 equinos, 11 ovinos, 25 suínos, 62 grupos de galinhas, 22 cães e 1 gato, residentes em 10 grandes propriedades privadas e 75 assentamentos rurais. Eimeria spp. estava presente em quase todas as espécies hospedeiras, com excessão de equinos, cães e gatos. Cistos de Giardia estavam presentes somente em bovinos. Nematodes foram encontrados em suínos, ruminantes e equinos em alta prevalência. Ancylostoma, Toxocara e Sarcocystis foram encontrados em cães. Galinhas foram encontradas com coccidia, Ascaridida e Capillaria spp.. Os parasitas encontrados podem causar má nutrição e problemas reprodutivos para seus hospedeiros. Medidas de prevenção e controle da dispersão de endoparasitas podem melhorar a condição de saúde pública, animal e ambiental nesta área.(AU)

Tick fauna of wild animals received and attended at the Santarém Zoological Park, western Park State, Brazil / Ixodofauna de animais silvestres resgatados e atendidos no Jardim Zoológico de Santarém, oeste do estado do Pará, Brasil

Ticks are known worldwide for parasitizing a number of wild hosts. However, few studies have been conducted on ticks in zoos in Brazil. The objective of the present study was to collect, identify, and report the parasitic tick fauna found on wild Amazon animals received and attended at the Santarém Zoological Park from September 2004 to September 2013. In all, 56 animals, including 26 mammals and 30 reptiles, were sampled, from which 1172 ticks were collected and identified, comprising 862 adults, 284 nymphs, and 26 larvae. Nymphs of Amblyomma geayi on three-toed sloth (Bradypus tridactylus), adults of Amblyomma longirostre on black dwarf porcupine (Coendou nycthemera), and nymphs of Amblyomma naponense on southern tamandua (Tamandua tetradactyla) were identified for the first time in the country in the present study. Although, the North region is the largest among the five Brazilian regions, this is the first study conducted with ticks and animals attended in a zoo in the Brazilian Amazon.

Carrapatos são conhecidos mundialmente por parasitar uma série de hospedeiros silvestres. No entanto, poucos são os estudos realizados com carrapatos em Jardins Zoológicos no Brasil. O presente estudo objetivou coletar, identificar e relatar a fauna parasitária de carrapatos de animais silvestres Amazônicos resgatados e atendidos no Zoológico de Santarém, durante o período de setembro de 2004 a setembro de 2013. Ao todo foram amostrados 56 animais incluindo 26 mamíferos e 30 répteis, no qual foram coletados e identificados 1172 carrapatos distribuídos em 862 adultos, 284 ninfas e 26 larvas. Foram identificados de forma inédita no presente estudo ninfas de Amblyomma geayi em preguiça-de-três-dedos (Bradypus tridactylus), adultos de Amblyomma longirostre em ouriço-cacheiro (Coendou nycthemera), além de ninfas de Amblyomma naponense em tamanduá-mirim (Tamandua tetradactyla) no país. Apesar da região Norte ser a maior entre as cinco regiões brasileiras, este é o primeiro trabalho realizado com animais atendidos em Zoológico utilizando os carrapatos como alvo de estudo na Amazônia nacional.

Tick fauna of wild animals received and attended at the Santarém Zoological Park, western Park State, Brazil / Ixodofauna de animais silvestres resgatados e atendidos no Jardim Zoológico de Santarém, oeste do estado do Pará, Brasil

Ticks are known worldwide for parasitizing a number of wild hosts. However, few studies have been conducted on ticks in zoos in Brazil. The objective of the present study was to collect, identify, and report the parasitic tick fauna found on wild Amazon animals received and attended at the Santarém Zoological Park from September 2004 to September 2013. In all, 56 animals, including 26 mammals and 30 reptiles, were sampled, from which 1172 ticks were collected and identified, comprising 862 adults, 284 nymphs, and 26 larvae. Nymphs of Amblyomma geayi on three-toed sloth (Bradypus tridactylus), adults of Amblyomma longirostre on black dwarf porcupine (Coendou nycthemera), and nymphs of Amblyomma naponense on southern tamandua (Tamandua tetradactyla) were identified for the first time in the country in the present study. Although, the North region is the largest among the five Brazilian regions, this is the first study conducted with ticks and animals attended in a zoo in the Brazilian Amazon.(AU)

Carrapatos são conhecidos mundialmente por parasitar uma série de hospedeiros silvestres. No entanto, poucos são os estudos realizados com carrapatos em Jardins Zoológicos no Brasil. O presente estudo objetivou coletar, identificar e relatar a fauna parasitária de carrapatos de animais silvestres Amazônicos resgatados e atendidos no Zoológico de Santarém, durante o período de setembro de 2004 a setembro de 2013. Ao todo foram amostrados 56 animais incluindo 26 mamíferos e 30 répteis, no qual foram coletados e identificados 1172 carrapatos distribuídos em 862 adultos, 284 ninfas e 26 larvas. Foram identificados de forma inédita no presente estudo ninfas de Amblyomma geayi em preguiça-de-três-dedos (Bradypus tridactylus), adultos de Amblyomma longirostre em ouriço-cacheiro (Coendou nycthemera), além de ninfas de Amblyomma naponense em tamanduá-mirim (Tamandua tetradactyla) no país. Apesar da região Norte ser a maior entre as cinco regiões brasileiras, este é o primeiro trabalho realizado com animais atendidos em Zoológico utilizando os carrapatos como alvo de estudo na Amazônia nacional.(AU)