The video, shot by his wife using a GoPro 3, shows the hefty fish as he nips at the man's flipper, tearing it off, and then goes straight for his catch with its powerful jaw. But, even if the diver wasn't familiar with that specific size of this type of fish, Goliath groupers have been known to roam western Atlantic waters near Florida.
On August 26th, Joshua Anyzeski caught the prohibited species, removing it from the water to take a picture. The picture circulated on social media, which tipped off officers with the Florida Fish and Wildlife Conservation Commission.
If you’ve never heard of the GoliathGrouper it is the largest of all the grouper species, and as we SE here it’s also a fish you never want to mess without in the ocean. The GoliathGrouper (formerly known as the Jewish) all-tackle world record was set back in 1961, in Fernanda Beach, FL…that fish was a colossal 680 POUNDS.
The Atlantic goliathgrouper or Tamara (Epimetheus Tamara), also known as the Jewish, is a large saltwater fish of the grouper family found primarily in shallow tropical waters among coral and artificial reefs at depths from 5 to 50 m (16 to 164 ft). Its range includes the Florida Keys in the US, the Bahamas, most of the Caribbean and most of the Brazilian coast.
On some occasions, it is caught off the coasts of the US states of New England off Maine and Massachusetts. In the eastern Atlantic Ocean, it occurs from the Congo to Senegal.
Young Atlantic Goliath groupers may live in brackish estuaries, oyster beds, canals, and mangrove swamps, which is unusual behavior among groupers. They may reach extremely large sizes, growing to lengths up to 2.5 m (8.2 ft) and can weigh as much as 360 kg (790 lb).
The world record for a hook-and-line-captured specimen is 308.44 kg (680.0 lb), caught off Fernanda Beach, Florida, in 1961. Considered of fine food quality, Atlantic goliathgrouper were a highly sought-after quarry for fishermen.
It is a relatively easy prey for spear fishermen because of the grouper's inquisitive and generally fearless nature. They also tend to spawn in large aggregations, returning annually to the same locations.
This makes them particularly vulnerable to mass harvesting while breeding. Until a harvest ban was placed on the species, its population was in rapid decline.
The fish is recognized as “vulnerable” globally and “endangered” in the Gulf of Mexico. The species' population has been recovering since the ban; with the fish's slow growth rate, however, some time will be needed for populations to return to their previous levels.
Goliath groupers are believed to be protogynous hermaphrodites, which refer to organisms that are born female and at some point in their lifespans change sex to male. Males can be sexually mature at about 115 centimeters (45 in), and ages 4–6 years.
In May 2015, the Atlantic goliathgrouper was successfully bred in captivity for the first time. Tidal pools act as nurseries for juvenile E. Tamara.
In tidal pools juvenile E.Tamara are able to utilize rocky crevices for shelter. Besides shelter, tidal pools provide E. Tamara with plenty of prey such as lobster and porcelain crab.
The Atlantic goliathgrouper has historically been referred to as the “Jewish”. It may have referred to the fish's status as inferior leading it to be declared only suitable for Jews, or the flesh having a “clean” taste comparable to kosher food ; it has also been suggested that this name is simply a corruption of jaw fish or the Italian word for “bottom fish”, Giuseppe.
In 2001, the American Fisheries Society stopped using the term because of complaints that it was culturally insensitive. Age, Growth, and Reproduction of Jewish Epimetheus Tamara in the Eastern Gulf of Mexico.
Pseudorhabdosynochus species (Monogenoidea, Diplectanidae) parasitizing groupers (Serranidae, Epinephrine, Epinephrine) in the western Atlantic Ocean and adjacent waters, with descriptions of 13 new species”. Wikimedia Commons has media related to Epimetheus Tamara.
Conservation physiology is a newly emerging discipline that examines the health status and physiological responses of wildlife to environmental disturbances in an effort to better understand factors contributing to population declines (1, 2). These assessments are crucial for conservation efforts, as wildlife are increasingly faced with such anthropogenic threats as climate change, habitat loss, pathogen introduction, contaminant exposure, and overexploitation (3 – 5).
Many of these perturbations are occurring at unprecedented rates and have already resulted in recognizable declines in individual and population health, making conservation and recovery efforts challenging (4, 5). Population-level health assessments involve a combination of physical and physiological examinations in an effort to establish “baselines” for hematological and biochemical analytes (2 – 4).
It is when shifts in these biomarkers occur that physiologically relevant alterations in population health in response to various stressors can be recognized (6, 7). Hematological and biochemical analyses can be useful in identifying stress, inflammation, diseases, and nutritional deficiencies, among many other underlying conditions or responses to physiological changes.
However, these diagnostic procedures are less frequently utilized in fishes compared to other vertebrate species, whether mammalian or not (9, 10). Baseline health studies in fishes have primarily focused on aquaculture species, including salmon , tilapia , sturgeon , and sea bass .
Reports of hematological and biochemical blood analytes are much less common for wild telecasts and elasmobranchs (15 – 19). These reference intervals can then be used as a basis for investigating spatial and temporal trends and for assessing population dynamics after potential environmental changes, stressors, or specific threats that might occur over time (5).
This species is the largest grouper in the Atlantic (reaching adult sizes of >2 m total length and 360–450 kg), long-lived (at least 37 years), charismatic, and it resides in waters of the western Atlantic Ocean, Gulf of Mexico, and Caribbean south to Brazil (21 – 24). GoliathGrouper are characterized as slow-maturing, protogynous hermaphrodites (21, 25) with known spawning locations in coastal waters of Florida in the southeastern United States (26, 27) and off the southern coast of Brazil (28).
They now show signs of population recovery off the coasts of Florida due to the closing of commercial and recreational fisheries from extractive exploitation but remain in low numbers elsewhere throughout their range. Several factors continue to cast doubt on whether full recovery has occurred and will be possible in Florida, including loss of mangrove habitat, destructive episodic events such as red tide and sudden cold exposure in winter months, and health and reproductive effects of high mercury concentrations in tissues and gonads (29 – 32).
Due to conservation efforts in the United States, Florida now serves as the center of recovery for the remaining GoliathGrouper population (32, 33). Baseline blood analyses can be used to assess sublethal impacts of environmental contaminants, but it is necessary to first establish reference intervals to gauge what is “physiologically normal” in these animals (34).
Therefore, the objectives of this study were to use non-lethal sampling methods (1) to establish reference intervals for hematological and plasma biochemical analytes, and report immune function, oxidative stress, and vitellogenin in mature males and females; (2) to evaluate total length, age, and sex in relation to blood analytes in juvenile and mature fish; (3) to assess analytes across sampled months in mature male and female fish; and (4) describe the typical light microscopy findings in liver and gill biopsies, including quantitative assessment of pigmented macrophages aggregates. This research was performed in accordance with institutional and national guidelines concerning the use of animals in research, and was approved by the Florida State University Institutional Animal Care and Use Committee (IACUC) (protocol #s: 1106, 1411, and 1718); Florida Fish and Wildlife Conservation Commission permits SAL-15-1244A-SRP, SAL-16-1244A-SRP, SAL-17-1244-SRP; and the National Oceanic and Atmospheric Administration permit F/SER24:PH.
This work was conducted throughout the coastal waters of Florida at depths between 10 and 35 m for adults and <0.1–2 m for juveniles. Juveniles were distinguished from mature adults based on sampling location, size, and age.
Several of us (26, 31, 35) developed non-lethal capture and sampling efforts that addressed federal and state laws requiring the release of this protected species alive and in good condition. On the deck of the boat, fish were immediately vented with a trocar and cannula to release gas from the swim bladder and to reduce pressure on internal organs, a wet towel placed over their eyes to protect from direct sunlight, a hose with flowing seawater placed over the gills for irrigation, and specular respiratory rate constantly monitored during the sampling procedure.
We did not see overt clinical reactions or evidence of pain caused by incisions or biopsies. From 2014 to 2017, adult GoliathGrouper were caught off the coasts of Florida, in the Gulf of Mexico and Atlantic Ocean.
In four cases, the same fish was caught twice over the 4 years of this study, from independent sampling trips. We treated recaptures of fish as independent samples because it was feasible that sufficient time had passed between captures to allow changes in their physiology.
Tissue and blood samples were collected immediately after fish were transferred to the deck of the boat, including (1) samples of dorsal fin rays (rays 6 and 7 excised at their base) for age determination ; (2) liver tissue and gill filaments for histopathological analysis; (3) gonad tissue for sex determination ; and (4) blood for analysis of various health variables. Dorsal fin rays were immediately placed on ice in the field and stored frozen at 20 °C until they were sent to D. Marie (University of Florida) for age determination <12 months after collection.
A small gill filament biopsy was taken near the edge using sterile surgical scissors. Following collection, tissues were immediately fixed in 10% neutrally-buffered formalin, and, following 24 h of fixation, the samples were rinsed and stored in 70% ethanol and shipped to Crowder Histology (Baton Rouge, Louisiana USA) for processing.
Processed tissues were embedded in paraffin, sectioned to 3–5 km thickness, and stained with hematoxylin and rosin (He) for viewing by light microscopy. Additional sections were stained with periodic acid Schiff (PAS) with and without diastase.
Gonad biopsies were obtained by inserting a polyethylene catheter into the conduct as per previously described techniques (27). Because this is a protogynous hermaphrodite, sexes included males, females, transitional, or unknown (where insufficient gonad tissue was collected).
At the site of blood collection, scales were removed using a blade and the entire area was repeatedly swabbed with sterile 70% isopropyl alcohol pads. Blood was collected via caudal venipuncture using either a 16- or 18-gauge heparin-coated needle and syringe (Supplemental Figure 1).
Collected blood was placed into 10 mL lithium-heparin coated BD Vacationers ® (Becton-Dickinson and Co., Franklin Lakes, New Jersey USA) and was carefully inverted to ensure adequate mixture with anticoagulant and subsequently placed over an ice bath in the field for up to 10 h until processing. Hemolytic was minimized by preventing direct contact between blood tubes and the ice.
Upon processing blood samples, a portion of whole blood was centrifuged (Fisher Scientific Model 228, Thermos Fisher Scientific, Waltham, Massachusetts USA) at 300 rpm for 10 min to harvest plasma. On the same day as sampling, heparinized whole blood was subsampled in micro capillary tube duplicates and sealed with Critical ® (Sherwood Medical Co., Demand, Florida USA).
After spinning, packed cell volume (PCV) was assessed as a percentage using a hematocrit micro capillary tube reader. Frozen plasma samples were sent to the University of Miami Avian and Wildlife Laboratory (UAW) for protein electrophoresis and quantification of vitellogenin (VG).
Two representative plasma protein electrophoretograms of a female and male GoliathGrouper indicating the 6 fractions of interest are presented in Supplemental Figure 2. Electrophoresis was carried out using split beta gels and the Spite 3,000 system (Helena Laboratories, Beaumont, Texas USA) to measure the concentrations of 6 fractions.
Measures of central tendency, range, and reference intervals (with 90% confidence intervals for upper and lower limits) for hematological, plasma biochemical, and plasma protein electrophoretic data for all adult Atlantic GoliathGrouper (Epimetheus Tamara) in conventional units. Vitellogenin was quantified using a grouper -specific VG ELISA kit, which employs the competitive enzyme immunoassay technique in the detection range of 60–1,200 NG/mL (MyBioSource, San Diego, California USA).
Vitellogenin was measured by adding standards or samples to appropriate wells containing horseradish peroxidase (Hip) conjugated-rabbit antibody. Plasma biochemical analytes were measured using the dry chemistry analyzer Videos 250XR (Or tho, Rochester, New York USA) at UAW and included alkaline phosphatase (ALP) aspartame aminotransferase (AST), blood urea nitrogen (BUN), calcium, cholesterol, creatine hospholipase (CK), creatinine, glucose, iron, lactate dehydrogenase (LDH), lipase, magnesium, phosphorus, potassium, sodium, total bilirubin, triglycerides, and uric acid.
Immune function, antioxidant capacity, and indicators of oxidative stress were quantified using plasma to measure activities of lysosome, glutathione peroxidase (GPX), superoxide dismutase (SOD), and concentrations of reactive oxygen and nitrogen species (ROS/RNS). Lysosome activity was analyzed using standard turbidity assays performed by Walsh et al. (37).
At UAW, GPX was measured using a commercially available glutathione peroxidase assay kit (Cayman Chemical Co., Michigan USA). A spectrophotometer was used to measure GPX activity following the oxidation of NADPH at 340 nm in this reaction.
SOD was quantified using a SOD assay kit (Cayman Chemical Co., Ann Arbor, Michigan USA), which detects superoxide radicals generated by Antoine oxidase and hypoxanthine through the use of a tetrazolium salt. SOD was measured as the proportional amount of enzyme needed to exhibit 50% disputation of the superoxide radical at an absorbance of 450 nm.
Concentrations of ROS/RNS were evaluated using an OxiSelectTM In Vito ROS/RNS Assay Kit (Green Fluorescence, Cell Bio labs, Inc., San Diego, California USA), following the manufacturer's instructions (38, 39). This assay universally measured ROS and RON species, which can include, among others, hydrogen peroxide, nitric oxide, peroxynitrite, and proxy radicals.
Additional analyses on liver tissue slides were conducted at Florida State University (Tallahassee, Florida, USA) to evaluate pigmented macrophages aggregates (PMA's), sometimes referred to as melano-macrophage centers, which are aggregates of highly pigmented phagocytes (40, 41). These were a prominent feature of liver tissue identified from preliminary histological analyses in this study.
To quantify area and count of PMA's, images were taken from a subset of the fish with liver biopsies collected (n = 55) using a Canon EOS equipped with a DSLR adapter for compound microscopes and Digital Photo Professional 4 software. For each prepared microscope slide, five images were taken of random and non-overlapping representative areas at each 100× and 400× total magnification.
Reference Intervals for hematology, plasma biochemistry, and protein electrophoresis data were calculated using Medical (Medical Software v.18.5, Os tend, Belgium) following the American Society of Veterinary Clinical Pathology reference interval guidelines using a combination of non-parametric and parametric methods, depending on sample size and distribution of the data (45). These same reference intervals were also converted to Standard International units (Supplemental Tables 1 – 3).
Measures of central tendency, range, and reference intervals (with 90% confidence intervals for upper and lower limits) for hematological, plasma biochemical, and plasma protein electrophoretic data for mature female Atlantic GoliathGrouper (Epimetheus Tamara) in conventional units. Measures of central tendency, range, and reference intervals (with 90% confidence intervals for upper and lower limits) for hematological, plasma biochemical, and plasma protein electrophoretic data for mature male Atlantic GoliathGrouper (Epimetheus Tamara) in conventional units.
We used canonical correlation analysis (CCA) as an exploratory analytical method to investigate the relationships between multivariate datasets of size (i.e., total length), age, and sex with health variables in all fish, including juvenile and mature fish and all sexes, to investigate potential morphometrical effects on blood analyte data. Linear regression analyses were carried out on each clinical and histological variable, including those where CCA was not performed, separately.
The only exception to this was for PMA count data with age and size, for which a GLM with family Poisson was conducted. Where assumptions were not met for linear regression analysis, packages COIN and Perm were used for permutation-based statistical tests with Monte-Carlo simulation as an alternative to classical procedures.
The categorical factor “sex” (male, female, transitional) was compared with separate clinical parameters using GLM with family Gaussian. The only exception to this was for PMA count data, for which GLM with family Poisson was conducted.
For these Gems, multiple comparisons were conducted using the glut function with packages em means and outcome to produce Turkey contrasts with adjusted p -values. The relationship between total length (TL, cm) and age (yrs) for GoliathGrouper was previously shown by Malinowski (31) to be strongly positively correlated (r 2 = 0.51, p < 2 × 10 16), but here we also compared size and age between males, females, and transitional using analysis of variance and Turkey contrasts with adjusted p -values.
To evaluate trends in blood analytes across months of sample collection for mature males and females, we used analysis of variance and Turkey contrasts with adjusted p -values after transforming (natural-log, square-root) any data that did need to meet parametric assumptions. For data that did not meet assumptions after transformation, we used Gems with the glut function for multiple comparisons.
For females, there was only one sample in October, and we decided to remove from this analysis because it could not be justifiably combined into the previous month. A total of 510 GoliathGrouper were caught, from which 195 blood samples were collected for hematological analysis.
Of the 139 samples used for blood plasma analyses, the major portion of this study, they were collected from different sexes and age groups (n female = 58, n male = 62, n transitional = 6, n unknown = 8, n juvenile = 5), with ages (yrs) ranging from 4 to 19 (median = 11, mean = 10.8, SD = 3.1) and total lengths (cm) from 57 to 219 (mean = 163.4, median = 165, SD = 27.5) (Supplemental Figure 3). A comparison of age and size between sexes for adults showed no difference in age, but a marginally significant difference in total length between males (mean = 163.8, median = 164, SD = 19.2) and females (mean = 173.0, median = 171, SD = 21.2) was observed, with females being larger (p = 0.03).
Conventional unit measures of central tendency, range, and reference intervals for all fishes, females only, and males only are reported in Tables 1 – 3, respectively. For plasma biochemical, immune system and oxidative stress analytes with too low of a sample size for reference intervals, descriptive statistics are reported in Table 4.
Descriptive statistics of immune and oxidative stress indicators, and vitellogenin, for adult (male, female, transitional) Atlantic GoliathGrouper (Epimetheus Tamara) that were not included as reference intervals. There were no significant correlations between hematology data, including PCV and reprogram, and age or total length (p > 0.05).
Thrombolytic numbers were determined to be adequate in all blood films evaluated, often with variably sized clumps that prevented a more detailed quantitative assessment. As described in Materials and Methods, CCA was not performed on PCV or VG due to few parameters and low sample size.
For plasma proteins, the first CCA (CCA1) was statistically significant (F = 2.257, p = 0.002) and strongly correlated with a canonical correlation coefficient (CCC) of 0.63 between plasma protein analytes and sex, age, and total length variables (Supplemental Table 5). Linear regression analysis revealed a number of significant results with protein fractions, which are detailed in Table 5.
Although sex had a fairly low canonical loading (loading strength = 0.11), we did find that males had significantly higher fraction 2 concentrations than females and that the concentration of this fraction for females was significantly negatively correlated with total length, with this female-only negative correlation (r 2 = 0.25) being stronger than with all sexes combined. The other sex-related result for proteins was that fraction five concentrations were significantly lower in transitional than both males and females.
PCV did not correlate significantly with total length or age and was not different between sexes (p > 0.05). Statistically significant plasma protein electrophoresis fractions and vitellogenin of the Atlantic GoliathGrouper (Epimetheus Tamara) categorized by sex (T, transitional; F, female; M, male) and by continuous variables of total length and age.
For females, protein fraction 1 significantly (p < 0.05) increased from May to July and August, and then decreased in September (Figure 1, Supplemental Table 4). Box plots showing significant (p <0.05) biochemistry analytes, plasma protein electrophoresis fractions, vitellogenin, and immune system and oxidative stress analytes for female Atlantic GoliathGrouper (Epimetheus Tamara) across sample collection months (May, July, August, September).
There was only one sample in October, and we decided to remove from this analysis because it could not be justifiably combined into the previous month. For males, protein fraction 3 was significantly (p < 0.05) lower from May and September, but August and October were not different (Figure 2, Supplemental Table 4).
Box plots showing significant (p < 0.05) biochemistry analytes, plasma protein electrophoresis fractions, and immune system and oxidative stress analytes for male Atlantic GoliathGrouper (Epimetheus Tamara) across sample collection months (May, August, September, October). Significant results for plasma biochemistry pairwise tests and linear regressions are detailed in Table 6.
Statistically significant plasma biochemistry analytes of the Atlantic GoliathGrouper (Epimetheus Tamara) categorized by sex (T, transitional; M, male; F, female) and by continuous variables of total length and age. Creatinine was significantly (p < 0.05) higher in males than females and transitional, and showed a negative relationship with total length and age.
There was also a significant (p < 0.05) negative relationship between creatinine and total length for males (r 2 = 0.18), with this correlation lower in comparison to all sexes combined (r 2 = 0.32). Lactate dehydrogenase, potassium, and uric acid all showed significant (p < 0.05) differences between sexes.
While glucose is an important metabolite that should be considered, we chose to exclude glucose data from reference interval development because samples were negatively affected by sample processing delay, which could not be avoided in this study. No other differences or relationships were observed for plasma biochemistry analytes, including ALP, AST, cholesterol, CK, magnesium, sodium, and total bilirubin.
For females, alkaline phosphatase, BUN, creatinine, iron, and lipase significantly (p < 0.05) decreased from May through September (Figure 1, Supplemental Table 4). For males, BUN and creatinine significantly (p < 0.05) increased from September to October, but were similar to each other in previous months (Figure 2, Supplemental Table 4).
As described in Materials and Methods, CCA was not performed on factors related to immune system and oxidative stress; however, pairwise tests and linear regressions were conducted and significant results are detailed in Table 7. Significant (p < 0.05) positive linear regressions, albeit fairly weak, occurred for lysosome with total length (r 2 = 0.13) and age (r 2 = 0.07).
Statistically significant immune system and oxidative stress analytes of the Atlantic GoliathGrouper (Epimetheus Tamara) categorized by sex (T, transitional; M, male; F, female) and by continuous variables of total length and age. For females, SOD significantly (p < 0.05) decreased from May through July and August, and then increased in September (Figure 1, Supplemental Table 4).
For males, lysosome significantly increased from August to September, but all other months did not differ from each other (Figure 2, Supplemental Table 4). Glutathione peroxidase significantly increased between September and October, but neither differed from May and August.
Age (range = 4–18 yrs) and sex were not significant factors in pigmented macrophages aggregate abundance. (A–C) Hepatocellular vacillation and variable pigmented macrophages appearance in livers of the Atlantic GoliathGrouper (Epimetheus Tamara).
(D) High magnification of a pigmented macrophages aggregate shows the presence of a trematode egg (arrow). (E) Variably sized globules of intracytoplasmic glycogen within hepatocytes stain deep magenta with PAS.
(F) Staining of the hepatocellular cytoplasm with PAS is lost after exposure to diastase, consistent with the presence of glycogen, while pigmented macrophages aggregates retain PAS staining, indicating the presence of glycoproteins, particularly Limousin. The most consistent microscopical change observed was mild to severe, diffuse, indistinct, cytoplasmic vacillation of hepatocytes, which were PAS positive and diastase sensitive, confirming glycogen content (n = 164/183, Figures 3A–F).
Rare to infrequent changes (Figures 4A–E) included minimal to mild lymphocytes hepatitis (n = 19/183), perirectal fibrosis (n = 9/183), lipid vacillation of hepatocytes (n = 7/183), mild, mixed perirectal and perivascular inflammation (n = 4/183), sinusoidal distension by round, clear, well-delineated spaces suggestive of gas bubbles that displace adjacent hepatocytes (n = 4/183, Figures 4A,B), and hepatocellular atrophy (n = 3/183). In many cases, liver sections contained rare to low numbers of mineralized granular (n = 72/183).
Trematode eggs consisting of a tan, refractive capsule and internal iridium were rarely to infrequently found within PMA's (n = 30/183, Figure 3D). Rare cross-sections of encysted nematodes ~120–150 km in diameter and featuring a ridged, refractive cuticle, coelomyarian musculature, pseudocoelom, lateral cords, intestine, and developing gonads were present within the hepatic parenchymal (n = 3/183, Figure 4C).
(A) Large, round, clear spaces consistent with air bubbles multi focally displace fibers in the heart of a fish that died during sampling. This study reports novel data on a suite of health variables for GoliathGrouper in Florida waters that provide an essential baseline “snapshot” and an important adjunct tool for assessing population recovery.
It also advances our knowledge about GoliathGrouper physiology and lays the groundwork for future research on the effects of various natural and human -induced stressors impinging on this species throughout its range where currently no such baseline health data exists. Given the abundance and novelty of data generated by this work, the focus of this current study lies on the animals and their intrinsic factors as no baseline health data of this kind exist to date on this or other GoliathGrouper populations throughout their range.
Future investigations into temporal, spatial, and various other aspects of this population will benefit from the baseline data presented herein. The influence of intrinsic (e.g., size, age, sex) and extrinsic (e.g., water temperature, habitat, diet, capture techniques) factors on various health variables is well-documented for many species .
Identifying and understanding the influence of these factors, which are more variable and less controllable in wild than managed individuals of the same species, are essential for accurate data interpretation. This is often the biggest challenge in wildlife health assessment studies, given the many logistical challenges in sample acquisition (e.g., stress from capture, effects from anesthesia if applicable) and potential confounding factors related to animal handling and sample collection.
In our study, animal capture and handling techniques, as well as analytical methodologies, were consistent for all analyses, thus minimizing possible variability from extrinsic factors. Therefore, we were able to focus interpretation of health assessment data of GoliathGrouper on morphometrics and time period of sampling, which are among the most important intrinsic and extrinsic factors, respectively.
Wildlife health assessments typically include the establishment of reference intervals for blood analytes, and often require partitioning of data into effects from factors such as habitat, season, spawning condition, length, age, and sex when sufficient numbers of samples are available (45, 55, 57). The observed significant difference between males and females in terms of total length, but not age, (Supplemental Figure 3) in this study population needs to be taken into account when considering the discussion of sex differences of various analytes.
Hematology data for GoliathGrouper showed more consistencies than differences across such intrinsic factors as length, age, and sex. PCV was comparable to other telecast species and fell mostly within reported ranges (62); however, the higher range in GoliathGrouper in this study exceeded the common upper limit of 45% PCV in telecasts, especially in males.
This suggests either a subtle sex difference and/or effects from stress during capture and handling (e.g., release of catecholamines resulting in hemoconcentration and swelling of RBC's) (55). Only three fish showed evidence of mild ethmoid regeneration, two of which with available PCV data were at the lower end of the reference interval (23 and 24%, respectively).
This observation may reflect a recent response to either anemia or transiently lower PCV. Regarding reprogram findings in GoliathGrouper, lymphocytes were the predominant WBC type, which is similar to numerous fish species reported in the literature (55, 62).
In addition, very low numbers of immature neutrophils were identified and hence are considered a normal finding in this species. In contrast to the consistencies with hematology data, plasma biochemical analytes showed several correlations in juvenile and mature fish and monthly trends in mature males and females allowing for considerations regarding some physiological aspects of this species.
However, since all correlations of blood analytes with morphometrics were overall weak in this study, these considerations are acceptable if interpreted with caution. This shows that this population with an average age of 10.8 yrs (range = 4–19 yrs) and an average total length of 163 cm (range = 57–219 cm) likely represents individuals in phases of somatic growth.
Albeit study animals were considered sexually mature, they presumably represent a younger population in the recovery stage, since the oldest GoliathGrouper reported to date was 37 yrs old, and, although the life span of GoliathGrouper remains unknown, this species is thought to live to much older ages (21, 32, 33). While this study provides a baseline for morphometrical data in this recovering population of GoliathGrouper, to see if these patterns and relationships hold in older and larger GoliathGrouper that reach their full growth and age potential, continued state and federal protection from lethal extraction of this species is necessary.
Several identified differences in plasma biochemical analytes with length, age and/or sex indicate potential significance regarding tissue growth and metabolic and/or nutritional changes in a growing, reproductively active population. These analytes include BUN and phosphorus, which were negatively correlated with length and age, possibly indicating a difference in feeding intensity or protein metabolism.
Higher BUN and creatinine in males compared to females and transitional, but lower uric acid in males compared to transitional suggest sex differences in muscle mass and/or protein/energy metabolism. This is further supported by variations in BUN, creatinine, and uric acid between adult males and females across sampled months.
Although fish primarily excrete nitrogenous waste as ammonia across their gills, urea is still present in all fish, if only comprising a low percentage of nitrogenous waste, and is often of research interest, along with creatinine, in diagnosing gill or liver disease (64, 65). Future GoliathGrouper research should consider these patterns along with environmental toxic ants in diagnosing disease or other negative health effects.
Creatinine, although similar to BUN in that it occurs in relatively small amounts in telecasts, can also be a good indicator of health and potential renal damage (64). The excretion and metabolism of nitrogenous wastes in fishes is still poorly understood and the clinical and physiological significance of these analytes still remains unknown (55).
Observed correlations, sex differences, and monthly trends of other analytes including calcium, cholesterol, iron, enzymes, and potassium may suggest subtle differences or variations in feeding frequency/fasting, diet, metabolic rates, reproductive physiology, or nutritional state of individuals prior to sample collection. In this same GoliathGrouper study population, a concurrent study found that males and females consumed the same prey but at different frequencies, and there was a lot of variation in stomach fullness, with many individuals having empty stomachs at time of capture (66).
Future research should directly consider diet and blood plasma analytes to better understand the relationship between these two factors. The protein electrophoretogram of the GoliathGrouper consistently provided 6 fractions across the analyses of all study samples.
Previously, 6 and 5 fractions have been described in Rainbow Trout Oncorhynchus my kiss (67) and KOI Cyprus cardio (68), respectively. Changes in acute phase proteins in fish with natural or experimental infection and with inflammatory processes have been documented (69) so it is likely that many of these fractions represent proteins, metabolites, and hormones that function similarly to their mammalian counterparts.
This conclusion is further supported by the observed variations in protein fractions across months in mature males and females. This is an interesting contrast to the positive correlation of lysosome with length and age, indicating a tendency to increase in growing GoliathGrouper, potentially suggestive of a greater capacity of the innate immune system and/or greater cumulative exposure to antigens as these fish age.
Sex differences were observed through differences of higher SOD in males compared to transitional, higher ROS/RNS species in males compared to females, and monthly trends of lysosome, SOD, and GPX in mature males and females, possibly suggesting variations in energy balance, diet, or an association with circulating reproductive proteins. Vitellogenin was positively correlated with length in females and negatively in males, with a substantial increase in mature females from respawning through spawning months, showing an overt sex difference in a maturing population with growth and sexual development.
Individuals for this health assessment study are well within the range of sizes and ages of transitional females observed for this population, which were shown in a previous study to range from 108 to 191 cm TL and 4 to 12 yrs of age (25). Similar to the pattern in VG, triglycerides were higher in females compared to males, suggesting a difference in circulating lipids and lipoproteins needed for vitellogenesis (73, 74).
Although there was overlap in plasma VG concentrations in males and females, these findings suggest possible utility of VG as a non-invasive biomarker for sex determination in consideration of size of fish and month of sampling given that analytical techniques are available via ELISA and proteins (72, 75). The baseline data presented herein may be useful for future studies of the effects of environmental estrogens and reproductive success (76, 77).
Hepatocyte morphology and appearance in fish liver tissue may vary considerably based on species, age, sex, season, nutritional status, and exposure to environmental pollutants (78). The histological changes described in this study reflect either typical, species-related observations, findings associated with life stage or active reproduction, or subclinical to clinically insignificant infectious and/or inflammatory processes.
The observed positive correlation of PMA % area and count with total length shows an increase as these fish grow and mature, presumptively from continuous active inflammation (e.g., antigen exposure) and/or metabolic changes over time. The main finding of prominent vacillation of hepatocytes was consistent with abundant, intracytoplasmic glycogen storage, which was confirmed by diastase sensitive PAS staining.
Excess energy intake beyond basal metabolism and other demands is often stored in fish hepatocytes as glycogen and/or lipid and utilized in fasting situations (79 – 81). Fish species physiology and diet components may predispose the accumulation of glycogen vs. lipid (79 – 81).
Glycogen accumulation is frequently seen in the hepatocytes of captive and occasionally wild fish, particularly trout and sea bass, which may reflect carbohydrate rich diets, minimal energy expenditure for foraging, and/or poor utilization of dietary carbohydrates (78 – 81). These changes were considered iatrogenic, and potentially due to capture methods bringing the fish up from depth, removal of surrounding pressure, and release of dissolved gases in the blood and highly vascularized tissues (82).
This last result is highly important for consideration of the health of GoliathGrouper in the current catch-and-release fishery that has grown into a major non-consumptive enterprise in Florida (32), in addition to optimizing fish capture techniques for health assessment studies. Although a catch-and-release fishery is a far better alternative to that of an extractive fishery, stress and other path-/physiological effects need to be further considered as this enterprise continues to expand, particularly related to barotrauma and how their dissolved blood gases shift out of solution, resulting in decompression-like embolisms as the fish is pulled rapidly to the surface.
For research purposes, and to better understand these effects while minimizing the stress on the animal, improvement to sampling techniques, as we have done in the past, would involve venting swim bladders mid-water prior to surfacing to minimize barotrauma, and/or using descending devices to safely return fish back to depth where expanded gases can contract . It adds to the otherwise sparse literature of hematological, biochemical, immune function, and oxidative stress data in free-ranging populations of marine telecast fishes.
It also demonstrates that non-invasive blood sampling provides an opportunity to obtain critical endpoint data and a vast amount of health information in a species in need of population and physiological monitoring. Additionally, our study adds to existing data on high muscle and liver tissue concentrations of mercury in this species that are among the highest of any telecast in the Atlantic or Gulf of Mexico (31).
The health indices reported herein will be critical for monitoring dynamics and demographics of the Florida population and for understanding physiological responses to various stressors and to a changing global oceanic environment specifically relevant to this population and associated organisms within their ecosystem (1). The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
NS: manuscript writing and editing, data interpretation, and blood film review. CC: blood plasma sample analyses, and manuscript writing and editing.
FC and CK: manuscript writing and editing, sample collection protocol, and general project oversight. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Although not an exhaustive volunteer list, we thank S. Adams, S. Bruegel, L. Buena, C. Compensate, J. Cu sick, R. Ellis, M. Palette, C. Label, T. Groan, A. Hal, M. Ruby, R. Johnson, B. Keller, J. Location, C. Match, R. McKenzie, D. McNutt, C. Mott, A. Nadella, M. Newman, K. Newton, N. Rag beer, T. Snow, C. Stalling, B. Salwar, B. Teak, O. Radio, M. Violin, K. Wall, and N. Enroll. We thank Rookery Bay National Estuaries Research Reserve for use of their facilities, which enabled sample collection in the Ten A Thousand Islands.
Deem SL, Harsh WB, Tasman W. Putting theory into practice: wildlife health in conservation. Clinical Methods for Assessment of the Effect of Environmental Stress on Fish Health.
Selected plasma biochemistry parameters in gilt head sea bream (Sparks aura ta) juveniles. Blood chemistry profile as indicator of nutritional status in European sea bass (Dicentrarchus Laura).
Candles K, Lie Ø, Wage R. Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon, Salmon solar. Hematology and plasma chemistry reference intervals for cultured tilapia (Oreochromis hybrid).
Knowles S, Huber TC, Smith SA, Basal R. Hematology and plasma chemistry reference intervals for cultured short nose sturgeon (Dispenser brevirostrum). Match MA, Arnold J, Jenkins E, Townsend H, Rosemary K. Determination of hematology and plasma chemistry reference intervals for 3 populations of captive Atlantic sturgeon (Dispenser oxyrinchus).
Plasma biochemistry reference values of wild bonnet head sharks, Smyrna bureau. Plasma biochemistry reference values of wild-caught southern stingrays (Asiatic Americana).
Haman Km, Norton TM, Thomas AC, Dove A. Baseline health parameters and species comparisons among free-ranging Atlantic sharp nose (Rhizoprionodon terraenovae), bonnet head (Smyrna bureau), and spiny dogfish (Squalls acanthus) sharks in Georgia, Florida, and Washington, USA. Satheeshkumar P, Nathan G, Senthilkumar D, Khan AB, Jeevanantham K. Comparative investigation on hematological and biochemical studies on wild marine telecast fishes from Cellar estuary, southeast coast of India.
Fabio F, Marriott S, Arturo F, Fiction G, Maggie C. Comparative study of the biochemical and hematological parameters of four wild Tyrrhenian fish species. Bullock LH, Murphy MD, Charles MF, Mitchell M. Age, growth, and reproduction of Jewish Epimetheus Tamara in the eastern Gulf of Mexico.
Saxony Y, Edmund A-M. Synopsis of biological data on the Nassau grouper, Epimetheus stratus (Bloch,1792) and the Jewish E. Tamara (Lichtenstein, 1822). NOAA Technical Report NMFS146, and FAO Fisheries Synopsis 157, Seattle, Washington (1999), p. 65.
Francesco J, Schwartz F. Jewish, Epimetheus Tamara, from North Carolina, with range correction and body comparisons. Regional Age Structure, Reproductive Biology and Trophic Patterns of Adult GoliathGrouper in Florida.
Buena LS, Bernini AA, Koenig CC, Coleman FC, Fracas MO, Late JR, et al. Evidence for spawning aggregations of the endangered Atlantic goliathgrouper Epimetheus Tamara in southern Brazil.
Malinowski C. High mercury concentrations in the Atlantic GoliathGrouper : spatial analysis of a vulnerable species. Koenig CC, Coleman FC, Klingon K. Pattern of recovery of the goliathgrouper Epimetheus Tamara population in the southeastern US.
The hematological assessment of the health of freshwater fish: a review of selected literature. Marie DJ, Parky DC, Koenig CC, Coleman FC, Schulz J, Frias-Torres S Evaluation of fin rays as a non-lethal aging method for protected goliathgrouper Epimetheus Tamara.
Walsh CJ, Legged SR, Carter BJ, Cole C. Effects of breve toxin exposure on the immune system of loggerhead sea turtles. Learn W, Gadupudi G, Well B, Simmons D, Olivier A, Robertson L. Progression of micronutrient alteration and hepatotoxicity following acute PCB126 exposure.
Paula M, Gauge E, Alloyed F, Leftover P, Stalls B. Ketone body therapy protects from cytotoxicity and acute liver failure upon Par deficiency. Melanomacrophage centers as a histological indicator of immune function in fish and other poikilotherms.
Mantra M, SAAF Default B, Pasquale JA, Girl L. Pigmented macrophages and related aggregates in the spleen of European sea bass dosed with heavy metals: ultrastructure and explorative morphometric analysis. Borucinska J, Koran K, Shacked M, Barker T. Melanomacrophages in three species of free-ranging sharks from the northwestern Atlantic, the blue shark Prince Laura (L.), the short fin make, Taurus oxyrhinchus Romanesque, and the thresher, Alias vulpine (Bonnaterre).
Friedrich KR, Hard KE, Freeman KP, Szladovits B, Walton RM, Barnhart Of, et al. ASVP reference interval guidelines: determination of DE Nova reference intervals in veterinary species and other related topics. Miffed AT, Ghelichpour M, Hosseini SM, Mini K. Hemolytic interference in measuring fish plasma biochemical indicators.
Quaglietta L, Mira A, Britain L. Extrinsic and intrinsic factors affecting the daily rhythms of a semiaquatic carnivore in a Mediterranean environment. Square AM, Rodríguez J, Guilty VM, Harcourt R, Hind ell M. Convergence of marine megafauna movement patterns in coastal and open oceans.
Satheeshkumar P, Nathan G, Kumar DS, Jagadeesan L. Hematology and biochemical parameters of different feeding behavior of telecast fishes from Cellar estuary, India. Fabio F. Fish hematology analysis as an important tool of aquaculture: a review.
Tavares-Dias M, Morals F. Hematological and biochemical reference intervals for farmed channel catfish. Reference intervals for hematological and plasma biochemical parameters in society sea bream juveniles (Sparidentex pasta, Valentines 1830).
Collins S, Kornberg A, Flores JM, Dombrowski DS, Herbart G. A comparison of blood gases, biochemistry, and hematology to geomorphology in a health assessment of pinkish (Lagoon rhomboids). Nelson K, Jones J, Jacobson S, Reimschuessel R. Elevated blood urea nitrogen (BUN) levels in goldfish as an indicator of gill dysfunction.
Malinowski C. Spawning Patterns, Trophic Ecology, and Toxicology: Conservation Related Research of an Iconic Reef Fish, the Atlantic GoliathGrouper. Mantra M, Britt D. Assessment of serum protein fractions in rainbow trout using automated electrophoresis and densitometry.
Respell S, Sullivan C. Gag (Mycteroperca microbes) vitellogenin: purification, characterization and use for enzyme-linked immunosorbent assay (ELISA) of female maturity in three species of grouper. Fremont L, Miami A. Biochemical analysis of vitellogenin from rainbow trout (Salmon Gardner): fatty acid composition of phospholipids.
Garcia-Garrido L, Muñoz-Chapuli R, DE Andres, A. Serum cholesterol and triglyceride levels in Scyliorhinus Caligula (L.) during sexual maturation. Application Mali TOF on protein identification of vitellogenin in giant grouper (Epimetheus lanceolatus).
Mills LA, Gutjahr-Gobell RE, Horowitz DB, Den slow N, Chow MC, Gerald EZ. Relationship between reproductive success and male plasma vitellogenin concentrations in runner, Tautogolabrus disperses.
Ends P, Reinsert S, Nashik S, Oliva-Teles A. Nutritional regulation of hepatic glucose metabolism in fish. Wolf JC, Baumgartner WA, Blazer VS, Camus A. Non lesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.
In: Leather land Of, Woo PT (eds) Fish Diseases and Disorders Vol. Descender devices are promising tools for increasing survival in deepwater groupers.