Projects

Universidade Federal do Amazonas (UFAM), Departamento de Biologia, ICB, 69077-000 Manaus, AM, Brasil

Biology Department, University of Puerto Rico - Rio Piedras (UPR-RP), San Juan, PR, 00931, Puerto Rico

Application of biotechnology to the study of economically important fishes of Amazônia

With the largest network of basins and the richest ichthyofauna on this planet, the commercial and artisanal fishery in Amazônia are extremely important activities in the region, both as a sources of income as well as ready source of protein for local peoples. Approximately 30 species of fishes are captured for commercial and subsistence purposes in Amazônia, generating millions of reais for various players. Large percentage of the species of fishes, such as the tambaqui, the pirarucu, the curimatã and the aruanã are fishery resources that are also exploited by other South American countries in the Amazon basin. The use scientific knowledge to manage and to guarantee the sustainable use of these resources is fundamental for both maintaining biodiversity, as well as for continued use of these resources as a source of income, work and source of food for the Amazonian region. Socio-economic benefits generated by the Amazonian fishery, and its long-term continuity depend directly on political decisions that guarantee the sustainable use of these resources and at the same time preserve these species and their genetic diversity. Today with the development and use of biomarkers, it is possible to obtain and elucidate fundamental information about genetic structure of populations, and especially those species with high commercial value for the Amazonian region. With this information public administrative sectors can implement better strategies of monitoring, management and conservation of Amazonian fisheries at various spatial and temporal scales . This project aims to provide this information, to build human resources for the next generation of management and conservation in Amazônia, and to develop a culture of collaboration between institutions, researchers and decision makers. This project is funded by CNPq.

Genetics, conservation and management of aquatic faunal of Amazônian várzea

This project is part of the research network entitled: Integrated management of fishery resources of Amazônian várzea: A comparative study of two regions: lower Amazonas and lower Purus

The goal of the present project is to characterize and delimit populations of key aquatic species using molecular techniques, with the goal of subsidizing the development of local strategies and policies for the conservation and management of aquatic fauna of the Amazonian várzea. A number of species with high commercial importance and value are already overexploited, and have experienced stock depletion in many areas. It is obvious that one of the great problems in effective management and conservation of the resources of the várzea is lack of fisheries data and of basic biological data on the organism to be managed, including genetics, as well as sociological and socioeconomic data on the riverine inhabitants. Understanding of the population genetic structure of each commercially important species is of importance for its management and effective utilization by riverine communities and commercial fishermen, and consequently for the long-term conservation of these species via participative action of the riverine communities which are principal actors in the process of sustainable development of Amazônia. Areas which will be studied in this project are the lower Amazonas region which already has participative and/or community management of aquatic resources, and the lower Purus River where community management is being implemented. Our study proposes to evaluate population genetic parameters and the distribution of genetic diversity over the Amazônian landscape of economically important species of fish, turtles and crocodilians, as well as of two key mammal species of the várzea. All these species have been and are exploited in various traditional and non-traditional forms, both legally and illegally. This includes even the two flagship mammal species, the Amazônian manatee whose meat is sold at clandestine markets, and the Amazônian pink dolphin which is slaughtered to be used as bait for necrophagous fish, and its eyes and genitalia are sold for makumba (voodoo) rituals. Aspects of phenotypic plasticity and adaptability will also be investigated. The genetic, morphometic and eco-physiological data will, together with other data collected by other sub-networks, form a substantial and integrated database for the sustainable management and conservation of aquatic resources of the várzea ecosystem of the Amazon basin. Funded by Brazilian Ministry of Science and Technology (MCT), United States Agency for International Development (USAID) and the G-7 countries, and administred by CNPq.

Application of biotechnology to the study of reproduction of the pirarucu (Arapaima gigas), an economically important fish of Amazônia

Lack of understanding of two important aspects of reproductive biology are major obstacles for captive reproduction of Arapaima gigas; if solved, applied research in fully artificial and semi-artificial reproduction of Arapaima gigas would be greatly facilitated. It is assumed but unknown that Arapaima are monogamous, however, whether egg clutches are fertilized by multiple males, or if these paternal mouthbrooders carry clutches of multiple females is unknown. Secondly, this species lacks sexual dimorphism, thus sex is impossible to determine in juveniles, and only with great difficulty in adults. Both aspects are important for successful aquiculture. Knowledge of reproductive strategies of Arapaima will facilitate providing proper reproductive conditions and towards maintenance of genetic diversity under artificial conditions, while being able to determine sex will significantly reduce operational costs of aquiculture facilities. The questions of sex and paternity may be resolved relatively simply with biotechnology. This project aims to use already developed microsatellite markers to analyze patterns of paternity in Arapaima gigas under different ecological conditions and in captive situations, and AFLP markers for the development of molecular sex markers. Funded by CNPq.

Multidisciplinary evaluation of the implementation of a system of lake management focusing on the conservation of the pirarucu (Arapaima gigas) in natural environments of the State of Amazonas

The pirarucu (Arapaima gigas) is one of the commercially most important fish species of the Brazilian Amazon. In the last two decades a disturbing situation was noticed with respect to natural stocks. There was a decrease in the volume of fish landed and in the average size of the fish landed which confirmed the suspicions of fisheries administrators and of scientists that pirarucu is being overexploited. In the Brazilian state of Amazonas, the commercial harvest of pirarucu is prohibited, but illegal commerce continues due to high demand for this species. It is evident that one of the big problems for the management of pirarucu in Amazônia is the lack of information about basic biology of pirarucu in the wild and the socioeconomic and cultural importance of this species for riverine fishing communities. Recently initiated studies in conjunction with local communities have started to generate important information about pirarucu, and its reproductive and fishery potential in areas of sustainable development (Mamirauá RDS - Amazonas state), and in communally administered areas (São Miguel Island, Ituqui Island and the Tapará region - Pará state). Although there exist legislative differences between these two types of regions, and many of these efforts are in their initial stages, the overall goal of these projects is to monitor the stocks of pirarucu, and to help the local communities to effectively participate in this monitoring by controlling access to lakes, by enforcing fishing regulations, but where permitted also to support sustainable commercialization of pirarucu products. Bio-ecological, social and economic components are tripod that determines the use of natural resources, and whose understanding and evaluation allows one to explain how the local population utilizes aquatic resources of the Amazon basin. This project seeks to supply knowledge for management and administration of fishery resources of Amazônia considering their distribution, abundance, population dynamics, and levels of genetic variability of exploited populations, and considering the various forms of local organization of the fishermen and their relationship with the market. Funded by FAPEAM.

Evaluation of the genetic variability of two species of ornamental fishes of the Mamirauá Sustainable Developmental Reserve: implications for sustainable management and conservation

Among the most commercially valuable fishes of the Mamirauá Sustainable Developmental Reserve are the aruanã (Osteoglossum bicirrhosum, family Osteoglossidae) and the acará-disco (Symphysodon aequifasciatus, family Cichlidae). Ornamental fishes are considered as one of the principal renewable aquatic resources in Amazônia, and they have a potential to serve as a model of sustainable management. Exploitation of these resources forms one of the principal means of subsistence of the riverine communities of the Mamirauá RDS, however, several species including the acará-disco have apparently suffered from over-exploitation in the past. Due to this state of affairs, harvest of ornamental fishes in the Mamirauá RDS is prohibited. In light of the potential importance of these resources to the riverine communities, it is necessary to evaluate the diversity, the ecology, the genetic structure, life cycle and natural densities of these fishes so that these commercially valuable species can be exploited sustainably. In the initial stage of this project, our group will collect aruanã and acará-disco from 11 micro-basins of the Mamirauá RDS in order to gather basic biological and ecological data on these species, and to evaluate genetic diversity within micro-basins, and genetic diversification between micro-basins. Additionally, the results of this study will tell us if formerly depleted stocks have recovered, and to what extent. This information will allow us to identify priority areas of conservation, areas suitable for exploitation, including rotational schemes of harvest, and to devise a management plan that will take all biological information into consideration. Projects such as this one seek to preserve the biological diversity of an area and at the same time promote a sustainable use of aquatic resources by local communities. Funded by the Mamirauá Civic Society.

Phylogeography and population genetic structure of Symphysodon Heckel, 1840, and the validity of the species S. discus Heckel, 1840 and S. aequifasciatus Pellegrin, 1904

The discus (Symphysodon spp.) are one of the most popular fish species in the specialized aquarium hobby, and also one of the commercially most valuable fish of the Amazon basin. In terms of export value, they form about 15% of the total ornamental fish export from Manaus estimated to be at over 100 million dollars per year, and are the most valuable species (cost/fish) exported. The natural distribution of the discus is the Amazon basin. Symphysodon discus (the Heckel discus) is found in the rio Negro basin, a major northern blackwater tributary of the Amazon; a population from the rio Trombetas, also a northern tributary of the Amazon, is also considered to be the Heckel discus. The subspecies Symphysodon discus willischwartzi (the pineapple discus) is found in the rio Abacaxis which is a minor tributary of the rio Madeira, itself a major southern muddy tributary of the Amazon. Other sections of the Amazon basin are occupied by the Symphysodon aequifasciatus. Two subspecies of S. aequifasciatus were described by Schultz: S. aquifasciatus haraldi, the ‘blue’ discus from Benjamin Constant found in upper the Amazon basin, and S. aequifasciatus axelrodi, the ‘brown’ discus from Belém found near the mouth of the Amazon River. The nominal subspecies S. aequifasciatus aequifasciatus, the ‘green’ discus, is found in the central portion of the Amazon basin. The distinction among the different subspecies of S. aequifasciatus is not always clear. Additionally, since Pellegrin, the original describer of S. aequifasciatus, never designated a holotype, and two of the three syntypes are from the geographical area of S. aequifasciatus haraldi and the third syntype is from the geographical area of S. aequifasciatus axelrodi, the subspecific descriptions cannot be considered valid. Most authors also do not recognize the subspecies S. discuss willischwartzi from rio Abacaxis, considering these fish to be hybrids, ‘cruzados’, between S. discus and S. aequifasciatus. To complicate matters even further, fishes with S. discus color pattern, a distinct black vertical bar in the center of its body, are also found in rio Jarí and occasionally also in rio Nhamundá. There is evidently a lot of phenotypic differentiation within both species, but traditional morphological methods cannot clearly partition any of this variation. Morphological differentiation between the two species is more easily defined, however. In addition to the distinctive black vertical band in the central portion of the body of S. discus, the two species differ in the mean number of scales (E series), and the mean number of dorsal rays and spines. However, there is no difference in other morphological characters, the two species readily hybridize in the aquarium and possibly also in nature. Furthermore, isozyme studies suggest there is as much genetic differentiation between different localities of S. aequifasciatus as there is between S. aequifasciatus and S. discus. To address these questions, we are using mitochondrial DNA sequence data to investigate phylogeography and population genetic structure of Symphysodon. Using these molecular sequence data, we will investigate if S. discus and S. aequifasciatus have their own separate and distinct evolutionary trajectories and are monophyletic, and thus are true species according to the phylogenetic species concept. We will also investigate if the large phenotypic variation generally classified as the ‘blue’, ‘green’ and ‘brown’ discus in S. aequifasciatus represents distinct evolutionary lineage, or if other morphologically or phenotypically unrecognized lineages exist in Symphysodon. Funded by DCG and IFS.

Phylogenetic systematics and evolution of complex life-histories in the Cyprinodontiformes

A multi-gene analysis is being used in an investigation of the phylogenetic relationships of fishes of the order Cyprinodontiformes. Preliminary analysis suggests a high concordance with global geological history. This hypothesis contradicts previously proposed phylogenetic hypothesis based on morphological data. Analysis of complex life-histories and morphologies suggests multiple, phylogenetically-independent evolutionary instances of diapausing eggs, annualism, copulatory organs, internal fertilization, live-bearing and maternal provisioning. The conclusions derived from the molecular hypothesis suggest many more phylogenetically-independent origins of complex life-histories and associated morphologies. While life-histories appear to repeatedly evolve in ecologically appropriate circumstances, similarities in morphologies and in the development of these morphologies begs the question of whether the underlying developmental genetic basis of complex life-histories and morphologies evolved convergently or due to parallelism. Funded by NSF.

Study and description of several new species of Rivulus from the Amazonas and Bolivar states of Venezuela

We are using a combination of morphometric, meristic, mitochondrial DNA and nuclear DNA data to revised and to describe new species of Rivulus from the Amazonas and Boliva states of Venezuela. Additionally, we are also using these data to obtain a better understanding of the phylogenetic relationships of members of this group of fishes, know as the Guyana Shield clade of Rivulus. This project is funded by the American Killifish Association.

Phylogeographic analysis of the genus Fluviphylax

We are using osteological, mitochondrial DNA and nuclear DNA data to investigate patterns and processes of diversification in the Neotropical genus Fluviphylax, the smallest Neotropical vertebrate genus. This project is funded by the Beca program, and the George Maier Fund from the American Killifish Association.

Quantitative genetic basis of vertebrate senescence and developmental diapause

Although senescence is inevitable, the time at which senescence occurs is controlled by both genetic and environmental factors. Senescence is a complex trait, and the biomedical importance of understanding senescence is likely to only increase as the human population ages. The group of fishes chosen as model organisms has 3-4 fold intraspecific variation in senescence, and over one order of magnitude intraspecific variation in length of developmental diapause. Species mature sexually in 3-4 weeks, and senesce in as little as six weeks. Large intraspecific variation in senescence of this vertebrate group puts this group of fishes at an advantage over non-vertebrate (e.g. Drosophila and C. elegans) as well as vertebrate (e.g. mouse) models for senescence. This group of small fishes also contains species that lay continuously developing eggs, as well as those that lay eggs undergoing up to three developmental diapauses. Our analyses suggest that the developmental diapause has five or more independent origins, and that senescence evolves rapidly and independently in response to environmental cues. The goal of this study is to perform a QTL analysis of senescence and developmental diapause in the fish super-family Aplocheiloidei. It is hoped that advances to public health will be generated by providing information on the complexity of senescence using a vertebrate model. As a result of this study we will discover how many chromosomal regions (and eventually genes) are involved in senescence, what are their interactions with each other and with environment, and if the underlying genetic basis of senescence in closely related organism is the same. Funded by NIH.

Tracking environmental change via developmentally complex life-listories

To investigate the response potential to environmental change, we are combining phylogenetics, selection experiments and life history data to study the evolution of developmental diapause and associated life history traits in response to environmental change in the fish super-family Aplocheiloidei. This super-family contains species that lay continuously developing eggs to those that lay eggs undergoing up to three developmental diapauses with a total duration of several years, and many species with more intermediate developmental conditions. Preliminary analyses suggest that the developmental diapause has evolved independently up to five times or more within this super-family; three times in South America and at least two times in Africa. Anatomical studies of eggs demonstrate that there are specific physiological and developmental adaptations associated with developmental diapause and that their complexity is proportional to the length of diapause. The specific goals of the current proposal are to: 1) characterize developmental diapause in representative subset of species in the super-family, 2) complete a phylogenetic analysis of the super-family and the order using molecular and morphological data, 3) combine the results of (1) and (2) to evaluate the patterns and origins of evolution of developmental diapause in the super-family, 4) use the comparative method to develop hypotheses for why and under what environmental conditions developmental diapause evolved, 5) do a laboratory selection experiment on diapause length, 6) measure diapause length in populations from recently antropogenically transformed areas and see if diapause length responded in accord with environmental change, and 7) generate a Geographic Information System (GIS) database to predict which environmental change scenarios will result in which life history strategies. Funded by NSF.

Phylogeography and population genetics of economically important species of ornamental fishes with differing life-histories

Ornamental fishes of the rio Negro region account for 90% of the ornamental fishes exported from the State of Amazonas, Brazil. It is estimated that at least 35 million individuals, over 85% of which are the cardinal tetra (Paracheirodon axelrodi), are exported annually from Manaus. Significant portion of the remaining 15% are the highly-valuable discus fish (Symphysodon discus and S. aequifasciatus). The export value of these fish is estimated to be over $4.5 million, and at least 10,000 Amazonians take part in the business. The ornamental fish industry depends on extractive activities which are promoted as a viable and sustainable alternative to deforestation of the region. However, history of extractive activities in the Amazon basin has repeatedly shown that in majority cases extractive activities are not sustainable. By analyzing the distribution of genetic diversity within and between collecting localities, and comparing these results among species, it will be addressed to what extent, if any, have populations of these heavily exploited aquarium fishes been impacted by commercial harvesting. These results will be used to access whether current levels of harvesting are sustainable. If harvested species show negative impact of overexploitation, we will work with management agencies to devise a sustainable harvest policy. Funded by IFS.

Genetics and molecular ecology of crocodilian populations of the Amazon

The principal objective of this project is to investigate the genetic structure and to access the genetic diversity of four species of crocodilians (or five species if Caiman yacare is considered a valid species) of Amazônia. Our specific objectives are: a) with respect to sampling with the whole of Amazônia 1) investigate how genetic variability of natural populations is currently distributed over the Amazonian geographic landscape; 2) investigate how patterns of historical and ongoing evolutionary processes are associated with the geography of the Amazon; 3) investigate if populations are in genetic equilibrium in light of the historical patterns of overexploitation; b) with respect to sampling within the rio Madeira 4) evaluate the species status of Caiman crocodilus, Caiman yacare, Paleosuchus trigonatus and Paleosuchus palpebrosus using morphological, ecological and genetic data; c) using all sampling 5) identify potential Evolutionary Significant Units (ESUs) or Management Units (MUs) to be used for the conservation and management of crocodilians. Funded by CNPq. Previous stages of this project were funded by WCS and TNC.

Comparative phylogeography of three species of freshwater rays of the family Potamotrogonidae: a test of hydrogeographic hypotheses of rio Negro

The family Potamotrigonidae is divided into three genera. The genera Paratrygon and Plesiotrigon are monotypic, while the genus Potamotrygon is represented by 16 to18 species. Species of this family occur in majority of the river systems of South America, with the exception of some Pacific drainages, and all coastal Atlantic drainages between the rio Parnaíba south to rio de la Plata separating Uruguay and Argentina. Given their wide distribution, the potamotrigonids are a highly suitable model for the study of historical biogeography of South America. In this particular study, we are estimating population genetic parameters and phylogeography of three species of Potamotrygon that occur in rio Negro. Using the observed patterns, we are testing hydrogeographic hypotheses as agents of diversification in this area of the Amazon basin. Formely funded by the PocketFund and the Beca program, and now by CNPq and The Explorer's Club.

Comparative population genetic study and paternity analysis of species of turtles of the genus Podocnemis of Amazônia using microsatellite and mitochondrial DNA markers

Quelonians are found in diverse localities including lakes, rivers, marshes, seas, forest and deserts. After IBAMA allowed captive breeding and ranching of some species (law 142/92 of IBAMA), it became apparent that very little was know about the population genetic structure of these species. Little is also known about paternity and population genetic structure within individual nests. Thus the principal goals of the present project is to characterize the population genetic structure of wild populations of three species of qualonians in the Amazon, the tracajá (Podocnemis unifilis), the irapuca (Podocnemis erythrocephala), and the iaças (Podocnemis sextuberculata) which occur within the Brazilian Amazon. The goals of this project is to contribute to the sustainable management and conservation of these species. Funded by CNPq.

Phylogeography of the Harpy Eagle (Harpia harpyja) in Brazil: genetic variability of representative individuals from three Brazilian biomes (Amazônia, Atlantic Rainforest and Pantanal)

In this project we study the evolutionary history, distribution and conservation of the top avian predator of Brazilian forests, and of all Latin American forests. Specifically our goals are to understand the phylogegraphic patterns of the Harpy eagle (Harpia harpyja) of Brazil. The Harpy eagle is a top level predator that lives in the upper canopies of New World forests from Mexico to Argentina, and is found in large areas of Brazil. It is the largest and most imposing falconiform bird of the world, has the largest tallons, and its back tallon (halox) can reach up to 10 cm. Globally, this species is considered to be threatened with extinction. In the Brazilian national list of threatened species, the Harpy eagle is not considered threatened, however, some state lists consider this species endangered. In the Brazilian Amazon this species is apparently abundant. The objective of this study is to infer the population genetic structure of the Harpy eagle in different forest habitats of Brazil, and to test if genetic differentiation exists between these discontinuous forests. The Harpy eagle is also relatively commonly confiscated by Ibama, the Brazilian fish and wildlife regulatory agency. Plans for release and reintroduction of confiscated animals is hampered by lack of knowledge of geographically compatible release sites. Similarly, it is often impossible to tell where confiscated animals came from, thus it is difficult to prosecute. Therefore the objectives of this study are also to generate a database which can be used by Ibama in its reintroduction and enforcement efforts. Funded by the Boticario Foundation and the Cleveland Metroparks Zoo through the Scott Neotropical Fund.

Population genetic analysis of the Bare-faced Tamarin (Saguinus bicolor): implications for conservation

Saguinus bicolor is the most endangered and least studies of all Amazonian primates. It appears to be largely restricted to forest remnants within the metropolitan Manaus. So far only a year long study of ecology and behavior of one group has been carried out. Scarce published information about this species refers primarily to geographic distribution which suggest that Saguinus bicolor is highly threatened with extinction since its habitat has been steadily encroached upon by the metropolitan expansion of Manaus in the last decades. In light of the threat of extinction of this primate due to habitat fragmentation and destruction caused by encroaching metropolitan Manaus, it is necessary to study various area of the biology of this species in order to formulate a conservation plan. Genetic studies utilizing microsatellite molecular makers are indispensable for the development of a conservation plan, and especially for those initiatives that will involve reintroductions and relocations of animals. Funded by MMA.

A population genetic analysis of social structure of the pink dolphin (Inia geoffrensis)

The pink dolphin Inia geoffrensis is a solitary species; at most mother offspring groups are observed. However, in several areas of the Amazon basin social groups of up to 12 individuals are known to exist. These groups exists in areas of high food abundance, and at least in some areas are directly associated with feeding by humans. The objective of this study is to investigate the population genetic structure of Inia geoffrensis of these groups, and to estimate relatedness of individuals within these groups. Do they form kin groups or are they just random assemblages of unrelated individuals? We are investigating these questions within the framework of the kin vs. group selection paradigm. Funded by the PocketFund.

Phylogeography and population genetic analysis of the Amazonian manatee (Trichechus inunguis)

In much of the Amazon basin, the Amazonian manatee Trichechus inunguis has been hunted to virtual extinction. The manatee has been protected since 1967, however, illegal hunting is practiced, and is especially prevelant in the Amazon River delta region. Manatees within the delta region appear to be further threatened through interspecific hybridization between T. inunguis and T. manatus. In addition to environmental education, the distribution and levels of genetic diversity, and the presence/absence and the frequency of hybridization will have important implications for conservation. This collaborative project is funded by the Beca program and CNPq.

Development of microsatellite molecular markers to investigate the population genetic structure of Cynoscion acoupa (pescada amarela)

The northern mangrove coast of Brazil comprises the Brazilian states of Amapá, Pará and Maranhão. This coast is over 2,500 km long and extends from the mouth of the rio Oiapoque south to the bay of São Marcos in Maranhão. Fishes from the coastal mangrove region form a principal component of the local fishery, and account for 58% of all fish landed. The most commonly landed fishes are those of the family Sciaenidae. In Brazil 61,400 tons of fish of this family are landed each year. This makes the Sciaenidae one of the economically most important groups of fishes. Cynoscion acoupa (pescada amarela) is one of the most explored sciaenid species with over 600 tons landed each year. We are therefore developing microsatellite markers to investigate genetic variability and population structuring of this species along the coast of Brazil. Funded by CNPq.

Genetic basis of dietary obesity

Genetic variation in response to high fat diets is important in understanding the recent secular trends that have led to increases in obesity and Type II diabetes mellitus. The examination of quantitative trait loci (QTL) both for obesity and diabetes-related traits and their responses to a high fat diet can be effectively addressed in mouse models systems, including the LGXSM recombinant inbred (RI) mouse strains. A wide range of obesity and diabetes-related traits were measured in animals from 16 RI strains with 8 animals of each sex fed a high or low fat diet from each strain. Marker associations were measured at 506 microsatellite markers spread throughout the mouse genome using a nested ANOVA. Locations with significant effects on the traits themselves and/or trait dietary responses were identified after correction for multiple comparisons by limiting the false detection rate. Non-syntenic associations of marker genotypes were common at QTL locations so that the significant results were limited to loci still significant in multiple QTL models. We discovered 91 QTLs at 39 locations. Many of these locations (n = 31) also showed genetic effects on dietary response, typically because the loci produced significantly larger effects on the high fat diet. Fat depot weights, leptin levels, and body weight at necropsy tended to map to the same locations and were responsible for a majority of the dietary response QTLs. Basal glucose levels and response to glucose challenge mapped together in locations distinct from those affecting obesity. These QTL locations form a panel for further research and fine-mapping of loci affecting obesity and diabetes-related traits and their responses to high fat feeding. Funded by NIH.