Larval digestive system ontogeny and early weaning in neon tetra Paracheirodon innesi.

The intensive culture of characid teleosts for ornamental trade is highly dependent on live feed organisms, particularly Artemia nauplii, to provide nutrition through the larval stage. Live feeds have inherent disadvantages relative to prepared microparticulate diets (MDs), specifically availability, labor and cost. In this research, the dependence of larval Paracheirodon innesi on live Artemia was confirmed via a nutritional trial. Next, digestive system ontogeny was characterized from the onset of exogenous feeding through metamorphosis. P. innesi exhibited an agastric larval stage, as well as low digestive enzyme activity at the onset of exogenous feeding followed by abrupt increases in trypsin, lipase and pepsin activity. Differentiation of the stomach, including gastric gland formation and production of neutral mucopolysaccharides, as well as the onset of pepsin activity, did not occur until 20 days post hatch (dph; 5.24 ± 0.20 mm). This shift from agastric to gastric digestive modes is indicative of a proliferation of digestive capacity and subsequent prey diversity in other fish species exhibiting similar altricial larval stages.Based on this information, different schedules for weaning from Artemia to a MD were evaluated. For P. innesi fed until 32 dph, weaning beginning at 12 dph and 17 dph resulted in similar survival to live Artemia (mean: 22.0 ± 1.7%), and the MD resulted in the lowest survival (0.8 ± 0.3%). These results indicate that weaning is possible prior to gastric differentiation, potentially resulting in the reduction of Artemia use in the larval culture P. innesi.

A scuticociliate causes mass mortality of Diadema antillarum in the Caribbean Sea.

Echinoderm mass mortality events shape marine ecosystems by altering the dynamics among major benthic groups. The sea urchin Diadema antillarum, virtually extirpated in the Caribbean in the early 1980s by an unknown cause, recently experienced another mass mortality beginning in January 2022. We investigated the cause of this mass mortality event through combined molecular biological and veterinary pathologic approaches comparing grossly normal and abnormal animals collected from 23 sites, representing locations that were either affected or unaffected at the time of sampling. Here, we report that a scuticociliate most similar to Philaster apodigitiformis was consistently associated with abnormal urchins at affected sites but was absent from unaffected sites. Experimentally challenging naïve urchins with a Philaster culture isolated from an abnormal, field-collected specimen resulted in gross signs consistent with those of the mortality event. The same ciliate was recovered from treated specimens postmortem, thus fulfilling Koch's postulates for this microorganism. We term this condition D. antillarum scuticociliatosis.

Cutaneous ulcerative lesions of unknown etiology affecting lionfish Pterois spp. in the Gulf of Mexico.

OBJECTIVE: Cutaneous ulcerative skin lesions in a complex of invasive Gulf of Mexico lionfish (Red Lionfish Pterois volitans, Devil Firefish P. miles, and the hybrid Red Lionfish × Devil Firefish) became epizootic beginning in mid-August 2017. Herein, we provide the first pathological descriptions of these lesions and summarize our analyses to elucidate the etiology of the disease. METHODS: We examined ulcerated and normal fish through gross pathology and histopathology, bacterial sampling, and unbiased metagenomic next-generation sequencing. We tracked prevalence of the disease, and we used biological health indicators (condition factor, splenosomatic and hepatosomatic index) to evaluate impacts to health, while considering sex and age as potential risk factors. RESULT: Typical ulcerative lesions were deep, exposing skeletal muscle, and were bordered by pale or reddened areas often with some degree of scale loss. Only incidental parasites were found in our examinations. Most fish (86%; n = 50) exhibited wound healing grossly and histologically, confirmed by the presence of granulation tissues. A primary bacterial pathogen was not evident through bacterial culture or histopathology. Metagenomic next-generation sequencing did not reveal a viral pathogen (DNA or RNA) but did provide information about the microbiome of some ulcerated specimens. Compared with clinically healthy fish, ulcerated fish had a significantly lower condition factor and a higher splenosomatic index. Disease prevalence at monitored sites through July 2021 indicated that ulcerated fish were still present but at substantially lower prevalence than observed in 2017. CONCLUSION: Although some common findings in a number of specimens suggest a potential role for opportunistic bacteria, collectively our suite of diagnostics and analyses did not reveal an intralesional infectious agent, and we must consider the possibility that there was no communicable pathogen.

Huffmanela cf. huffmani (Nematoda: Trichosomoididae) infecting swim bladder, peritoneum, and gonad of variable platyfish, Xiphophorus variatus (Cyprinodontiformes: Poeciliidae) and eastern mosquitofish, Gambusia holbrooki (Poeciliidae) in Florida; taxonomy, phylogenetic analysis, and pathological changes.

Variable platyfish, Xiphophorus variatus (Meek, 1904) (Cyprinodontiformes: Poeciliidae) and eastern mosquitofish, Gambusia holbrooki Girard, 1859 (Poeciliidae) from earthen ponds in west central Florida were examined for parasitic infections. At necropsy, we observed myriad nematodes (adults and eggs), which we identified as Huffmanela cf. huffmani, infecting the swim bladder, gonad, and visceral peritoneum. Nucleotide sequences (small subunit ribosomal DNA, 18S) of H. cf. huffmani from variable platyfish and eastern mosquitofish were identical; likewise for newly obtained 18S sequences of Huffmanela huffmani Moravec, 1987 from the swim bladder of red breast sunfish, Lepomis auritus (Linnaeus, 1758) (Centrarchiformes: Centrarchidae) and warmouth, Lepomis gulosus (Cuvier, 1829) from the San Marcos River (type locality for Huffmanela huffmani Moravec, 1987), Texas. The sequences of H. huffmani and H. cf. huffmani differed by 7 (1%) nucleotides. Pathological changes comprised proliferation of the tunica externa of the swim bladder in low-intensity infections in addition to inflammation, proliferation, and tissue necrosis of swim bladder, peritoneum, and gonad in high-intensity infections. The lesion was severe, affecting the cellular constituents of the swim bladder wall and reducing the size of the swim bladder lumen; potentially reducing swim bladder physiological efficiency. The present study is the first record of a freshwater species of Huffmanela Moravec, 1987 from beyond the San Marcos River, first record of a species of Huffmanela from a livebearer, first nucleotide sequences and phylogenetic analysis for Huffmanela, and first evidence that an infection by a species of Huffmanela causes pathological changes that could impact organ function.

Preliminary Investigations into Use of Metomidate for Euthanasia of Ornamental Fishes.

The goals of this study were to investigate the potential use of metomidate for one-step euthanasia of ornamental fish species representing commonly sold families in the ornamental fish trade and to determine a baseline euthanasia dose for most species tested. Metomidate hydrochloride, a rapid-acting, water-soluble, nonbarbiturate hypnotic related to etomidate, was tested at various concentrations and durations for euthanasia of species representing the following freshwater and marine aquarium fish families: Apogonidae, Ariidae, Blenniidae, Callichthyidae, Characidae, Cichlidae, Cyprinidae, Gobiidae, Gyrinocheilidae, Loricariidae, Melanotaeniidae, Osphronemidae, Pimelodidae, Poeciliidae, Pomacentridae, and Pseudochromidae. Fish in each trial were euthanized as a group (n = 1). Most groups contained 10-12 fish. Some higher doses required buffering. Euthanasia was considered successful if all fish in each group did not recover after 6 h in unmedicated water. All species immediately lost buoyancy and equilibrium, dropping to the tank bottom within 1 min and ceasing ventilation typically within minutes. However, reactivity to vibration, sound, or touch was noticeable for varying time periods afterward (8-66 min), so an additional 30 min of exposure after cessation of reactivity was included as part of the protocol. Although some species (Neon Tetras Paracheirodon innesi and Australian Rainbowfish Melanotaenia australis) were euthanized at a concentration of 40 mg/L metomidate for a total exposure time of 38 min, most species tested were successfully euthanized with metomidate at a concentration of 100 mg/L, with total exposure times ranging from 35-96 min. A few catfish species (Otocinclus sp. and Bronze Corydoras Corydoras aeneus) were refractory and recovered after 100 mg/L. However, Otocinclus sp. were successfully euthanized at 250 mg/L, and Bronze Corydoras were euthanized at 1,000 mg/L. Further studies are needed to provide additional data for these and other species and families, for different water chemistry conditions, and for various biological factors to fine-tune dosing regimens.

Complete genome sequences of infectious spleen and kidney necrosis virus isolated from farmed albino rainbow sharks Epalzeorhynchos frenatum in the United States.

The genus Megalocytivirus includes viruses known to cause significant disease in aquacultured fish stocks. Herein, we report the complete genome sequences of two megalocytiviruses (MCVs) isolated from diseased albino rainbow sharks Epalzeorhynchos frenatum reared on farms in the United States in 2018 and 2019. Histopathological examination revealed typical megalocytivirus microscopic lesions (i.e., basophilic cytoplasmic inclusions) that were most commonly observed in the spleen and kidney. Transmission electron microscopic examination of spleen and kidney tissues from specimens of the 2018 case revealed hexagonally shaped virus particles with a mean diameter of 153 ± 6 nm (n = 20) from opposite vertices and 131 ± 5 nm (n = 20) from opposite faces. Two MCV-specific conventional PCR assays confirmed the presence of MCV DNA in the collected samples. Full genome sequencing of both 2018 and 2019 Epalzeorhynchos frenatus iridoviruses (EFIV) was accomplished using a next-generation sequencing approach. Phylogenomic analyses revealed that both EFIV isolates belong to the infectious spleen and kidney necrosis virus (ISKNV) genotype within the genus Megalocytivirus. This study is the first report of ISKNV in albino rainbow sharks.

Blood Analyte Changes of Wild-Caught Adult Almaco Jack in Response to Acclimation to Recirculating Aquaculture Systems and Hyposalinity Treatment.

Despite the utility of blood analyte evaluation as a diagnostic tool to assist in monitoring the health of marine fishes, baseline data are often lacking for many commercially important finfish species. The objective of this study was to compare hematology and plasma chemistry data for adult wild-caught Almaco Jack Seriola rivoliana at time of capture and again following a period of acclimation to a recirculating aquaculture system and hyposalinity treatment. A total of 30 clinically healthy adult fish were caught via hook and line in the eastern Gulf of Mexico, approximately 120 mi offshore from Madeira Beach, Florida. Blood was collected from a subset of these fish (n = 13) immediately after capture and again at 16 weeks postcapture from another subset (n = 12) following a 45-d antiparasitic hyposalinity treatment. A 19% increase in fish body weight was observed during the study period (16 weeks) and no overt health issues or mortality were noted. Compared to fish that were sampled immediately following capture, several significant differences (P < 0.05) were observed. Absolute white blood cells were lower in captive held fish, suggesting biological variation, antigenic stimulation in wild fish, and/or immunosuppression associated with stress in captive held fish. Lower sodium, chloride, and calculated osmolality indicate osmoregulatory adjustments following the hyposalinity treatment by 16 weeks postcapture. Other observed plasma biochemical differences presumptively reflect dietary and/or environmental changes, or physiological variation following acclimation to captive culture conditions. This study reports baseline blood analyte data of wild-caught Almaco Jack and documents hematological and plasma biochemical responses to their new environment as captive broodstock. Baseline hematological and plasma biochemistry data obtained during this study are the first reported for this species.

Gastrointestinal entamoebiasis in captive anurans in North America.

Infections with Entamoeba spp. are recognized as a cause of clinical disease in many species including humans and reptiles; however, cases in amphibians are under-reported. Investigation of a mortality event among a captive population of Cranwell's horned frogs Ceratophrys cranwelli at a production facility in Florida, USA, revealed that deaths were due to the newly described Entamoeba species CT1. Infection caused severe necroulcerative gastroenterocolitis with a predilection for the colon. To date, this Entamoeba species has only been described in invasive cane toads Rhinella marina in Australia. Retrospective screening of archived anuran cases from a zoological pathology service identified 8 cases from captive populations that had histological evidence of gastrointestinal entamoebiasis. Molecular characterization was positive in 3 cases. Two cases, 1 in a Puerto Rican crested toad Peltophryne lemur and 1 in an Amazon milk frog Trachycephalus resinifictrix, showed 100% homology to E. ranarum and 1 case in a White's tree frog Litoria caerulea showed 100% homology to Entamoeba sp. CT1. This is the first report of novel Entamoeba sp. CT1 being associated with clinical disease in anurans within North America and also the first report of this Entamoeba species causing disease within managed collections as far back as 2003.

Hematology and Biochemistry Panel Reference Intervals for Captive Saddleback Clownfish Amphiprion polymnus and Tomato Clownfish A. frenatus.

Reference intervals of blood analytes are important diagnostic tools that clinicians use as a basis for making clinical decisions and that have been documented for many fish species. Although the effects of dietary supplementation on hematology and blood biochemistry variables in clownfish have been shown, there are no published reference intervals to date for routine hematology and blood biochemistry panels for these popular cultured marine fish (including Amphiprion spp.). The objective of this study was to establish de novo reference intervals for selected hemogram data and, using whole-blood analysis, commonly utilized chemistry analytes in two species of aquacultured clownfish: the Tomato Clownfish Amphiprion frenatus and Saddleback Clownfish A. polymnus. This study is based on blood samples from 25 clinically normal Tomato Clownfish and 38 clinically normal Saddleback Clownfish from two clownfish production facilities in Florida. Guidelines by the American Society for Veterinary Clinical Pathology were followed to generate robust reference intervals from the data collected. We report descriptive statistics and reference intervals for several hematology (n = 12) and biochemistry (n = 8) analytes using a small sample size and a small amount of blood per sample. The blood analyte data from this study are comparable with hematology and biochemistry data that have been reported in other teleost species. These results provide baseline health data for clownfish species that can be used in various clinical settings while also serving as a springboard for further research studies.

Reference Intervals for Blood Analytes of Adult Aquarium-Housed Russian Sturgeon Acipenser gueldenstaedtii.

Russian Sturgeon Acipenser gueldenstaedtii are an important, critically endangered, roe-producing species. Despite a wealth of knowledge pertaining to other members of family Acipenseridae, there is very limited published information regarding baseline blood analytes in Russian Sturgeon. The objectives of this study were (1) to establish reference intervals for a suite of hematological and biochemical data and (2) to compare plasma chemistry data to two point-of-care (POC) cartridges, tested on the VetScan iSTAT 1 analyzer, that use heparinized whole blood for the assessment of clinically normal, aquacultured adult Russian Sturgeon sedated with eugenol (AQUI-S 20E) at a single institution. Reference intervals are reported. The calculated hematocrit measured by the POC analyzer tended 4-5% lower than the spun packed cell volume, confirming the importance of spun packed cell volume as a reliable measurement of red blood cell mass. Various analytes, notably whole-blood urea nitrogen, glucose, sodium, total carbon dioxide, chloride, ionized calcium, and anion gap, were significantly different by both POC cartridges. This study successfully produced reference intervals for blood analytes in adult Russian Sturgeon under managed care and creates a foundation for future studies into the effects of extrinsic and intrinsic factors and variations of analytical methodologies on blood analytes in this species.

Characterization of a novel picornavirus isolated from moribund aquacultured clownfish.

Over the last decade, a number of USA aquaculture facilities have experienced periodic mortality events of unknown aetiology in their clownfish (Amphiprion ocellaris). Clinical signs of affected individuals included lethargy, altered body coloration, reduced body condition, tachypnea, and abnormal positioning in the water column. Samples from outbreaks were processed for routine parasitological, bacteriological, and virological diagnostic testing, but no consistent parasitic or bacterial infections were observed. Histopathological evaluation revealed individual cell necrosis and mononuclear cell inflammation in the branchial cavity, pharynx, oesophagus and/or stomach of four examined clownfish, and large basophilic inclusions within the pharyngeal mucosal epithelium of one fish. Homogenates from pooled external and internal tissues from these outbreaks were inoculated onto striped snakehead (SSN-1) cells for virus isolation and cytopathic effects were observed, resulting in monolayer lysis in the initial inoculation and upon repassage. Transmission electron microscopy of infected SSN-1 cells revealed small round particles (mean diameter=20.0-21.7 nm) within the cytoplasm, consistent with the ultrastructure of a picornavirus. Full-genome sequencing of the purified virus revealed a novel picornavirus most closely related to the bluegill picornavirus and other members of the genus Limnipivirus. Additionally, pairwise protein alignments between the clownfish picornavirus (CFPV) and other known members of the genus Limnipivirus yielded results in accordance with the current International Committee on Taxonomy of Viruses criteria for members of the same genus. Thus, CFPV represents a proposed new limnipivirus species. Future experimental challenge studies are needed to determine the role of CFPV in disease.

First Record of A Species of Mermithidae Braun, 1883 Infecting a Decapod, Palaemon paludosus (Palaemonidae).

The hemocoel of 26 of 30 (86%) eastern grass shrimps, Palaemon paludosus (Gibbes, 1850) (Decapoda: Palaemonidae), captured during June 2017 from several freshwater lakes near Leesburg and Lake Kissimmee, Florida, was infected by juveniles of a nematode species (Mermithidae sp.). Some infected eastern grass shrimps were preserved whole for histopathology, whereas others were dissected to excise parasitic juvenile nematodes, and still others were kept alive in glass aquaria such that post-parasitic (emerged) juvenile nematodes could be opportunistically observed alive and ultimately preserved. Parasitic and post-parasitic juvenile nematodes had cuticle cross-fibers, cephalic papillae, cup-shaped amphids, a horn-shaped vagina, a trophosome, and a caudal appendage, which collectively diagnosed them as Mermithidae sp. They differed from those of nematomorphs (Nematomorpha) by lacking 4 giant cells anteriorly, tegumental bristles, scale or plate-like areoles, a bifurcate or trifurcate posterior end, and an anus. A phylogenetic analysis of the small subunit rDNA (18S) that included all of the available mermithid sequences corresponding to morphologically diagnosed specimens recovered Mermithidae sp. within the clade of mermithids and sister to Ovomermis sinensis Chen, Jian, and Ren, 1991 . This is the first record of a mermithid infection in a decapod and first report of a mermithid infection in an aquatic crustacean from North America (another mermithid infects a terrestrial isopod there). The high prevalence of infection and the multiple geographic localities harboring infected eastern grass shrimps indicated that these infections were not spurious. Because no other decapod is confirmed as a mermithid host, we suspect that these specimens likely represent a new species with a life cycle worth studying, since none for a mermithid involving a decapod nor a crustacean has been elucidated to date. We also provide a table of all mermithid and nematomorph infections in crustaceans.

Phylogenomic characterization of red seabream iridovirus from Florida pompano Trachinotus carolinus maricultured in the Caribbean Sea.

Between 2010 and 2016, six mortality events were observed in Florida pompano (Trachinotus carolinus) maricultured in the Dominican Republic. Histopathological examination and conventional PCR confirmed a megalocytivirus (MCV) infection in each case. Subsequently, next-generation sequencing and phylogenomic analyses confirmed that MCV DNA was present in the infected pompano tissue samples from 2010, 2014, and 2016, and each was determined to be red seabream iridovirus (RSIV). Annotation of the RSIV genome sequences identified 121 open reading frames, and BLASTN analysis revealed the highest nucleotide sequence identity (> 99%) to a RSIV clade 1 MCV isolated from a moribund red seabream (Pagrus major) maricultured in Japan. These cases represent the first fully sequenced RSIV genomes detected outside of Asia and are the earliest reports of MCV infections in Florida pompano. This recent geographical expansion of RSIV warrants further attention to determine its potential economic and ecological impact.

Phylogenomic characterization of two novel members of the genus Megalocytivirus from archived ornamental fish samples.

The genus Megalocytivirus is the most recently described member of the family Iridoviridae; as such, little is known about the genetic diversity of this genus of globally emerging viral fish pathogens. We sequenced the genomes of 2 megalocytiviruses (MCVs) isolated from epizootics involving South American cichlids (oscar Astronotus ocellatus and keyhole cichlid Cleithracara maronii) and three spot gourami Trichopodus trichopterus sourced through the ornamental fish trade during the early 1990s. Phylogenomic analyses revealed the South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) possess 116 open reading frames each, and form a novel clade within the turbot reddish body iridovirus genotype (TRBIV Clade 2). Both genomes displayed a unique truncated paralog of the major capsid protein gene located immediately upstream of the full-length parent gene. Histopathological examination of archived oscar tissue sections that were PCR-positive for SACIV revealed numerous cytomegalic cells characterized by basophilic intracytoplasmic inclusions within various organs, particularly the anterior kidney, spleen, intestinal lamina propria and submucosa. TSGIV-infected grunt fin (GF) cells grown in vitro displayed cytopathic effects (e.g. cytomegaly, rounding, and refractility) as early as 96 h post-infection. Ultrastructural examination of infected GF cells revealed unenveloped viral particles possessing hexagonal nucleocapsids (120 to 144 nm in diameter) and electron-dense cores within the cytoplasm, consistent with the ultrastructural morphology of a MCV. Sequencing of SACIV and TSGIV provides the first complete TRBIV Clade 2 genome sequences and expands the known host and geographic range of the TRBIV genotype to include freshwater ornamental fishes traded in North America.

Odontomas in two long-finned ocellaris clownfish ( Amphiprion ocellaris).

Two adult long-finned ocellaris clownfish ( Amphiprion ocellaris) from 2 different aquaculture facilities were examined at the University of Florida, Tropical Aquaculture Laboratory for oral masses. Incisional biopsies were obtained from the masses under anesthesia, and histologic examination revealed both to be odontomas. Most benign odontomas are classified as hamartomas (disorganized proliferations of tissues found normally at the site of origin). Odontomas have previously been reported in wild teleost fish in association with viral infections and pollution.

Identification of Francisella noatunensis in novel host species French grunt (Haemulon flavolineatum) and Caesar grunt (Haemulon carbonarium).

Francisella noatunensis is an emerging pathogen of fish that has been isolated from several cultured species worldwide. Here presented is a case involving several hundred marine grunts that were caught near the Florida Keys for display in public aquaria. These fish were maintained in a recirculating system where they began to experience mortalities approximately two weeks post-stocking. Postmortem examination revealed disseminated systemic granulomatous disease most severely affecting spleen and kidney. Splenic and renal tissue homogenates inoculated in modified Thayer Martin agar media yielded colonies consistent with F. noatunensis 4 days post inoculation. Bacterial colonies and tissues were confirmed positive after real-time PCR amplification of the intracellular growth loci gene (iglC) specific for F. noatunensis subspecies orientalis. Consequently, multiple novel host species for this pathogen were identified, including the French grunt (Haemulon flavolineatum) and the Caesar grunt (Haemulon carbonarium).

New disease records for hatchery-reared sturgeon. II. Phaeohyphomycosis due to Veronaea botryosa.

A series of fungal cases in hatchery-reared juvenile and young adult Siberian sturgeon Acipenser baerii and white sturgeon A. transmontanus occurred at production facilities in Florida and California, USA, respectively. Affected fish exhibited abnormal orientation and/or buoyancy, emaciation, coelomic distension, exophthalmos, cutaneous erythema, and ulcerative skin and eye lesions. Necropsies revealed haemorrhage throughout the coelom, serosanguinous coelomic effusion and organomegaly with nodular or cystic lesions in multiple organs. Fungal hyphae were observed in 27 fish (24 A. baerii and 3 A. transmontanus) via microscopic examination of tissue wet mounts and on slides prepared from colonies grown on culture media. Histopathological examination of these infected tissues revealed extensive infiltration by melanised fungal hyphae that were recovered in culture. Phenotypic characteristics and sequencing of the fungal isolates with the use of the internal transcribed spacer region and 28S rRNA gene confirmed the aetiological agent as Veronaea botryosa. To our knowledge, this is the first documentation of V. botryosa infection in fish, although melanised fungi of the closely related genus Exophiala are well-known pathogens of freshwater and marine fishes.

Development of a nonlethal health assessment for wild Red Drum using a health index.

Nonlethal methods are needed to assess the health of wild fish and quantify the robustness of the broader population. Results could be used to indicate exposure to various stressors, such as contaminants, infectious disease, external parasite loads, and fishing pressure, to monitor changes in fish population health over time. The wild Red Drum Sciaenops ocellatus population in the Kennedy Space Center Reserve of Merritt Island National Wildlife Refuge was used to develop a protocol to define the health of free-ranging fish using nonlethal techniques. This health index incorporated morphometric measurements, weight, an evaluation for external parasite fauna, notation of physical deformities, and the presence of lesions. A total of 126 adult Red Drum were collected using hook-and-line angling during prespawning (May), spawning (September and October), and postspawning (December) periods. All fish were released alive back into their environment. The nonlethal health assessment scored fish in the "healthy" range of the health index during the prespawning and spawning periods. Fish caught during the postspawning period scored slightly below this range. Parasite load contributed to the depressed score during the postspawning period. Fish collected in all sampling periods were rated on average as "excellent" for condition factor, which suggests that the sampled population in the reserve were thriving.