Name: necropsy-based wild fish health assessment
Uploaded: 2018-09-11T13:00:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Necropsy-based Wild Fish Health Assessment at JoVE.com

This work was funded by the U.S. Geological Survey's Ecosystems (Chesapeake Bay Environments and Fisheries) and Environmental Health (Contaminants Biology) programs and the West Virginia Department of Natural Resources. Use of trade names is for identification purposes only and does not imply endorsement by the U.S. government.

Methods described here have been approved by the Leetown Science Center&#39;s Institutional Animal Care and Use Committee.
1. Fish Collection
<ol>
	<li>Collect live fish with a minimum of stress. Use boat or backpack electrofishing, hook and line or nets.</li>
	<li>Hold fish in live wells or aerated containers until sampling. 
	NOTE: The American Fisheries Society has published a number of guides for fish collection, handling and anesthesia/euthanasia26,27,28. Wear gloves when handling fish.</li>
</ol>
2. Fish Necropsy
<ol>
	<li>Euthanize a fish.
	<ol>
		<li>Place fish in the anesthetic until opercular movement ceases and the fish loses equilibrium. After another 2&#8211;10 min the fish will be euthanized; however, this may also vary by species. 
		NOTE: Fish can be euthanized with a number of anesthetics (see Table of Materials for most commonly used). The method of euthanasia will depend on the laboratory measurements that will be conducted on tissues collected29.</li>
	</ol>
	</li>
	<li>Measure biometric characteristics.
	<ol>
		<li>Weigh the fish to the nearest gram.</li>
		<li>Measure the fish length to the nearest millimeter.
		<ol>
			<li>Measure the total length from the tip of the snout with the mouth closed to the end of the tail when pinched together.</li>
			<li>Measure the fork length from the fork in the tail to the tip of the snout, and the standard length from the tip of the snout to the end of the body (beginning of the tail).</li>
		</ol>
		</li>
		<li>Calculate the condition factor using the following formula: 
		Condition factor = (total body weight - gonad weight)/total length3. 
		Note: Gonad weight is subtracted from the total body weight since gonads can contribute significantly to the total body weight, particularly in prespawn female fish.</li>
	</ol>
	</li>
	<li>Obtain a blood sample. 
	NOTE: Blood is most commonly taken from the caudal vein but can also be withdrawn from the dorsal aorta or by cardiac puncture30.
	<ol>
		<li>Extract a peripheral blood sample from the caudal vein with a 22 or 23 G needle on a 1 to 5 mL syringe, depending on size of the fish. Insert the needle anterior to the caudal area below the lateral line (Figure&#160;1A and 1B). Angle it upwards until hitting the spine and then withdraw slightly. The vein is ventral to the overlying spine. 
		NOTE: If blood smears will be made or serum is required, no anticoagulant is used. In most cases, plasma will be collected and, hence, an anticoagulant such as sodium heparin, EDTA or lithium is used to coat the needle and syringe and is also in the blood collection tube (e.g., vacutainer).</li>
		<li>Remove the needle and place into a sharps disposal container prior to putting the blood into the collection tube. 
		NOTE: Blood can be held on ice but depending on subsequent analyses should be centrifuged as soon as possible30.</li>
		<li>If nuclear abnormalities or differential blood counts will be evaluated, immediately place a drop of blood on duplicate clean glass microscope slides. Back a second slide at a 45&#176; angle into the drop, which is then drawn across the surface by capillary action. Allow to air-dry31.</li>
		<li>Centrifuge blood at 1,500&#8211;2,500 x g for 15 min to sediment the cells. Remove plasma/serum with a sterile transfer pipet, aliquot into cryogenic vials, and store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57946/57946fig1.jpg" /> 
Figure 1: Obtaining a blood sample from a fish.&#160;(A) A recently euthanized fish is laid on its side and the lateral line located. (B) A needle is inserted ventral to lateral line (arrow), angled upward until needle touches the backbone. It is then slightly withdrawn, and suction initiated to withdraw blood. <a href="https://www.jove.com/files/ftp_upload/57946/57946fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Conduct a necropsy-based health assessment on each fish. 
	NOTE: A number of publications illustrating and describing lesions and abnormalities are available32,33,34,35.
	<ol>
		<li>Document external abnormalities including lesions on body surface and fins (Figure 2), eyes and gills (Figure 3), external parasites such as leeches (Figure 2D), grubs or trematode metacercarial cysts (Figure&#160;2D, 3B) and gill parasites (Figure 3D). Document type, location and size of observed abnormalities on data sheets, as well as photographically, if possible.</li>
		<li>Open the abdominal cavity (Figure 4A) using a scissors by cutting from the anal area to the operculum and then removing the flap of muscle to expose the internal organs. 
		NOTE: If anterior kidney will be collected for immune function (see step 5 below) or samples collected for bacteriology or virology, the external body surface should be disinfected with 70% alcohol and those samples should be obtained before the necropsy performed. If tissues are only being used for visual observations, plasma analyses and histopathology sterile technique is not necessary.</li>
		<li>Document internal abnormalities (Figure 4) including general or focal discolorations of the various organs (Figure&#160;4B-4D), presence of raised areas (Figure 4E), cysts, parasites, and size abnormalities (enlarged, atrophied).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57946/57946fig2.jpg" /> 
Figure 2: Examples of visible lesions observed on body surface and fins of fish.&#160;(A) A small, slightly eroded lesion (arrow) on the lateral body surface. (B) A large reddened area (arrow) involving the caudal body surface. (C) Raised, black lesions (arrows) on the body surface and fins. (D) Leeches (white arrow) and small black spots (black arrows) on the fin. Scale bar = 3 mm. (E) A raised, multilobed, pale lesion (arrow) on the body surface. <a href="https://www.jove.com/files/ftp_upload/57946/57946fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57946/57946fig3.jpg" /> 
Figure 3: Examples of visible lesions of the gills and eyes of fish.&#160;(A) A pale area (arrow) within the lens of an eye. Scale bar = 5 mm. (B)&#160;White cysts (white arrows) and small black spots (black arrows) caused by trematode parasites on the operculum covering the gills (a). Scale bar = 1 cm.&#160;(C) A pale, eroded area (arrow) on the gill (a). Scale bar = 5 mm.&#160;(D)&#160;A gill that has been removed showing parasites (arrows) attached to the gill filaments. Scale bar = 2 mm. <a href="https://www.jove.com/files/ftp_upload/57946/57946fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57946/57946fig4.jpg" /> 
Figure 4:&#160;Examples of a necropsy and internal abnormalities of fish.&#160;(A) During a necropsy the fish is cut open (along the white arrow) and a flap of muscle (black arrow) removed to expose the gonad (a) and the spleen, being held by forceps&#160;and scissors. (B)&#160;Mottled liver (a), testes (b), intestine surrounded by adipose fat (c) and stomach (d). Scale bar = 5 mm. (C)&#160;Liver (a) with a dark red area (arrow), ovary (b) and intestines (c). Scale bar = 5 mm. (D)&#160;Liver with greenish discolored areas (arrows). Scale bar = 1 cm. (E) Example of a normal (a) and abnormal (b) testes with raised nodules. Scale bar = 1 cm. <a href="https://www.jove.com/files/ftp_upload/57946/57946fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Obtain hepatosomatic index (HSI).
	<ol>
		<li>Remove liver by severing the hepatic artery and the connective tissue of the anterior end. Gently lift out while trimming adhesions and other connections to intestine and adipose fat. Take care to not puncture the gallbladder. Weigh the liver. 
		NOTE: Some fish, such as cyprinids, do not have a discrete liver but rather hepatic tissue wrapped around intestines and other organs. For these species, it may not be possible to obtain liver weights.</li>
		<li>Calculate hepatosomatic index (HSI) using the formula: 
		HSI=liver weight/total body weight</li>
	</ol>
	</li>
	<li>Calculate the gonadosomatic index.
	<ol>
		<li>Remove the gonads and weigh it.</li>
		<li>Calculate gonadosomatic index (GSI) using the formula: 
		GSI = gonad weight/total body weight</li>
	</ol>
	</li>
</ol>
3. Preserve Tissues for Microscopic Pathology
NOTE: A number of fixatives including 10% neutral buffered formalin and Z-fix, a formalin-based fixative with zinc, can be used for preservation of tissue in the field. The latter is preferred if methods such as&#160;in situ&#160;hybridization or fluorescent antibody staining may be used.
<ol>
	<li>Carefully cut but do not pull out tissue samples. Keep individual tissue pieces &#60;2 cm in size and &#60;5 mm thick for proper fixation. As a rule of thumb, use approximately 10x more fixative by volume than the tissue for proper preservation. Place all tissue samples from one fish in the same leak-proof container of the appropriate size, depending on the size of fish being sampled.</li>
	<li>Place pieces of any external abnormalities in the fixative container. Also, include an adjacent piece of normal tissue. 
	NOTE: Improper handling such as compression or other mechanical damage, long exposure to air or sunlight, and freezing can cause artifacts.</li>
	<li>Cut at least five 3&#8211;4 mm thick pieces of liver from various regions and place into the fixative container. Include normal and abnormal areas, if observed.</li>
	<li>Depending on size, place a whole gonad or multiple pieces along one gonad into the fixative container.</li>
	<li>Place either whole organs, if small, or pieces of all other organs (spleen, anterior and posterior kidney, gills, heart, intestine and stomach) in the fixative container. If abnormal tissue is observed, preserve an adjacent piece of normal tissue as well.</li>
</ol>
4. Remove the Otoliths for Age Analyses
NOTE: Age can be an important variable in fish disease/fish health studies. While a number of structures, including scales and spines, have been used for age determination, most studies comparing structures have found the otoliths to give the best results36,37. Teleost fishes have three pairs of otoliths - lapillus, sagitta and asteriscus. Generally, the sagittal or lapillus otoliths are collected for aging although that may vary by species. Removal and aging techniques have been previously described38.
<ol>
	<li>Cut through the gill isthmus, and bend the head back. Strip away connective and muscular tissue around the inferior portions of the neurocranium to locate prootic bullae, a raised bony area.</li>
	<li>Score or cut with bone cutters and crack to expose the otoliths. They can be seen with the naked eye.</li>
	<li>Place otoliths in a labelled vial or a coin envelope and store at room temperature until analyzed for age by counting the rings or increments38. If placing in a vial, open cap once returned to the laboratory and allow to thoroughly dry prior to storage.</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57946/57946fig5.jpg" /> 
Figure 5: Removal of otoliths.&#160;(A)&#160;The isthmus is cut and the connective tissue and muscle pulled away to expose the base of spine and neurospinal area. (B)&#160;The bone is cracked to expose the otoliths. (C)&#160;Lapillar otoliths are removed. <a href="https://www.jove.com/files/ftp_upload/57946/57946fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Obtain Tissue for Immune Function Assays
NOTE: The anterior kidney is the major hematopoietic organ, the source of lymphocytes and macrophages for functional assays, and must be removed aseptically if cells will be cultured for functional assays, such as mitogenesis, phagocytic and killing ability of macrophages39,40.
<ol>
	<li>Spray the external surface of the fish with 70% ethanol. Use sterile scissors, a scalpel and forceps to open the abdominal cavity and remove the anterior kidney tissue, which is a dark red organ located anterior to the swim bladder.</li>
	<li>Place the anterior kidney sample in media (e.g., Leibovitz&#39;s L-15) to keep the cells alive. Homogenize kidney samples with a sterile hand-held tissue grinder (e.g., Tenbroeck tissue grinder) into single cell suspensions. Hold on wet ice until returned to the laboratory.</li>
</ol>
6. Preserve Tissue for Nucleic Acid Analyses
NOTE: If downstream molecular analysis will be conducted, such as gene expression using transcript abundance41&#160;or quantitative PCR42&#160;(polymerase chain reaction), place the pieces of tissue to be assessed in an appropriate preservative (e.g., RNAlater stabilization solution) as soon as possible.
<ol>
	<li>For RNA preservation, place two to three small (2&#8211;3 mm) pieces in the appropriate preservative at a 10:1 ratio of preservative volume to tissue. 
	NOTE: Samples should be shielded from sunlight or excessive heat and transported on wet ice.</li>
	<li>For DNA preservation, place two to three small pieces of tissue into 95% ethanol (10:1 ethanol to tissue by volume). Then hold the samples on wet ice and then store at -20 &#176;C.</li>
</ol>

The authors have nothing to disclose.

The necropsy-based assessment of fish health can be utilized on any fish species for which the investigator has an understanding of the normal appearance of both external and internal structures. Using a standardized approach allows for comparisons between sites and species as well as seasonal and temporal changes in a population. The findings can be used to identify effects associated with point and nonpoint sources of contaminants and to inform management actions. It can also be used to track improvements once management actions are initiated. The methodology can be modified to augment the documentation of visual external abnormalities in a variety of ways. Assessments, based only on visual observations, can be non-lethal, relatively inexpensive and data can be generated quickly for a large number of individuals. Consequently, they can be useful for exploratory or initial assessments, to monitor change over time or in combination with other indicators. If the length and weight of fish are measured during visual observations, the condition factor can also be calculated. Although assessments based only on visual observation do not provide information on cause or associated risk factors, long term trends of certain skin abnormalities45 and biometric parameters46 have indicated improvement in some areas associated with water quality improvements.
The necropsy-based assessment provides more information as internal organs are also examined and other condition factors such as hepatosomatic index and gonadosomatic index can be calculated. Goede and Barton22 developed a field necropsy method that included blood parameters, biometric factors, the percentage of abnormalities, and index values for specific abnormalities. A refinement of the method included a severity rating for some variables that allowed for calculation of a health assessment index that could be compared statistically23. This health assessment index has been used in regional site comparisons23,47,48 and in combination with other biological indicators including plasma and histopathological analyses in the U.S. Geological Survey&#39;s Biomonitoring of Environmental Status and Trends Program evaluating potential effects of contaminant exposure in large rivers nationwide49,50,51. A Fish Disease Index based on externally visible diseases and parasites, visible liver neoplasms and other histopathologically detected liver lesions has been developed and used extensively in the North Sea, Baltic Sea, and off Iceland. This index was found to be an important tool as an ecosystem health indicator52.
There are some critical factors in conducting the necropsy-based assessment on fish. First, assessments must be conducted on fish immediately after death. Changes in organ color and consistency can occur fairly rapidly after death. Additionally, some parasites may leave the host soon after death. Second, it is important to know what is normal for the species of interest. For instance, some fish normally have fatty and consequently, pale livers, while for most species a pale liver would be abnormal. It is also important to recognize seasonal changes that naturally occur. Some fish will have color changes or develop breeding tubercles during the spawning season.
The limitations of the necropsy-based assessment as a method for fish health assessment include the inability to 1) consistently identify the &#34;cause&#34; of specific lesions and 2) identify effects that may not be visible to the naked eye. These drawbacks can be overcome with the addition of histopathology, molecular or cultural identification of pathogens and parasites, and gene expression. For instance, a &#34;tumor&#34; or raised lesion (swelling) may be actual neoplasia or it may be a parasite, inflammation, edema or hyperplasia (increase in number of normal cells), caused by chemical exposure, infectious agents or other irritants. As shown in the representative results, definitive tumor or neoplasia diagnosis requires microscopic pathology to identify the lesion type and severity (i.e., benign or malignant). Assessment of white sucker external &#34;tumors&#34; by visual observation overestimated the prevalence, particularly at the reference site. Many of the raised lesions were not neoplasms but rather hyperplastic lesions. It is currently not known whether these hyperplastic lesions are pre-neoplastic. Conversely, the observation of raised nodules in the liver significantly underestimated the prevalence of liver neoplasms. Hence, collection of tissue for microscopic pathology was necessary to adequately address the potential for delisting.

Human activities have numerous adverse effects on aquatic environments. Fish inhabit various water bodies that the human population recreates in and often uses as a drinking water source and hence are important indicators of the health of the aquatic environment. Wild fish that live and reproduce in a particular habitat are exposed throughout their lives to various stressors including pathogens, parasites, poor water quality and chemical contaminants. Thousands of chemicals enter our waterways through industrial and human wastewater, suburban/urban stormwater and agricultural runoff. These complex mixtures of chemicals can have additive, synergistic or antagonistic effects on exposed organisms1,2,3. In addition, other environmental stressors such as elevated nutrients, elevated temperature, low dissolved oxygen or fluctuating pH can exacerbate the effects of chemical contaminants4,5. Environmental stressors can also influence infectious disease outcomes directly by increasing the number of infectious agents6, increasing the virulence of opportunistic pathogens7 or suppressing the immune response and disease resistance of the host8,9,10. For these reasons, there is increasing interest in biological or adverse effects monitoring11,12,13,14, utilizing fish and other aquatic organisms to identify populations and ecosystems at risk.
Adverse effects monitoring utilizes biomarkers at various levels of organization, from the organismal to the subcellular or molecular, to identify sublethal effects which may influence populations and be indicative of exposure to various stressors. Indicators at the organism level include visible abnormalities and conditions. Condition indices based on length and weight are calculated to evaluate the well-being or fitness of fish populations. The most common is Fulton&#39;s condition factors (K) = (weight/length3) 15. Another indicator is the presence of visible abnormalities. A variety of methods have been used in individual studies and monitoring programs to assess, document, and evaluate visible abnormalities. Assessment based only on external abnormalities, i.e., the proportion of individuals with disease, fin damage, tumors and skeletal anomalies, is one of the metrics for the index of biotic integrity (IBI) that evaluates community health16. A similar assessment termed DELTs (deformities, erosions, lesions, tumors) has also been used to evaluate the health of fish communities17. However, these methods only assess external visual abnormalities and not internal lesions or early sublethal indicators.
Necropsy-based assessments include external and internal observations and allow for the measurement of additional condition indices. Hepatosomatic index (liver weight/total body weight) has also been used as an indicator of fitness or energy reserves15 for which a higher index value indicates healthier fish. However, a number of studies have shown that hypertrophy or an increase in liver size occurs due to exposure to various contaminants metabolized by the liver18,19,20. In this case a higher index would be indicative of exposure to certain chemical classes. The gonadosomatic index (gonad weight/total body weight) is another condition index directed toward reproductive health21. Observations made during the necropsy-based assessment can be used to compare the prevalence of individual lesion types or percentage of normal individuals. However, they can also be used in a more quantitative health assessment22,23.
The standardized necropsy-based assessment described here can be used to augment the grossly visible assessment in multiple ways depending on the question(s) to be answered, expertise and other available resources. Our routine approach is to collect biometric data (length, weight, liver weight, gonad weight), blood for plasma/serum analyses, document external and internal visible abnormalities, preserve pieces of organs for microscopic analyses and collect otoliths for age analyses. The necropsy-based assessment plus age analysis and histopathology of various organs, allows for the calculation and comparison of various condition indices, prevalence of visible abnormalities, as well as microscopic tissue changes, by sex, age, site and sampling period. Additional tissue collections can be made for many other analyses including electron microscopy, bacteriology, virology, parasitology and chemical concentrations. These methods can also be part of more in-depth analyses used to diagnose the cause of fish kills24 or mortalities of captive fishes25. Methods for collection of tissue for two additional analyses, gene expression and functional immune analyses are illustrated.

<table><tbody><tr><td>Folding tables</td><td>Any</td><td></td><td></td></tr><tr><td>Folding chairs</td><td>Any</td><td></td><td></td></tr><tr><td>Dissecting boards</td><td>Any</td><td></td><td></td></tr><tr><td>Measuring board (mm)</td><td>Any</td><td></td><td></td></tr><tr><td>Battery powered scale (g) for fish weight</td><td>Any</td><td></td><td></td></tr><tr><td>Battery powered scale (mg) for organ weights</td><td>Any</td><td></td><td></td></tr><tr><td>Dissecting forceps</td><td>Any</td><td></td><td></td></tr><tr><td>Bone cutters</td><td>Any</td><td></td><td></td></tr><tr><td>Scalpel and blades</td><td>Any</td><td></td><td></td></tr><tr><td>Disposable gloves</td><td>Any</td><td></td><td></td></tr><tr><td>Buckets</td><td>Any</td><td></td><td></td></tr><tr><td>Leak-proof Nalgene bottles (250 mL)</td><td>ThermoFischer Scientific</td><td>02-924-5C</td><td></td></tr><tr><td>Vacutainer tubes with sodium heparin</td><td>ThermoFischer Scientific</td><td>02-689-6</td><td>For blood collection</td></tr><tr><td>Disposable&amp;nbsp; 3 mL syringes with 23 G needle</td><td>ThermoFischer Scientific</td><td>14-826-11</td><td></td></tr><tr><td>1 &amp;ndash; 2 mL cryovials</td><td>Any</td><td></td><td>Used for plasma and RNAlater samples</td></tr><tr><td>Invitrogen RNAlater Stabilization solution</td><td>ThermoFischer Scientific</td><td>AM7021</td><td></td></tr><tr><td>Z-Fix Formaldehyde Zinc fixative</td><td>Anatech LTD</td><td>SKU-174</td><td></td></tr><tr><td>Tricaine-S (MS-222)</td><td>Syndel USA</td><td></td><td>fish anesthetic</td></tr><tr><td>Coin Envelopes</td><td>Any</td><td></td><td>for otoliths</td></tr><tr><td>Pencils and pens</td><td>Any</td><td></td><td></td></tr><tr><td>70% alcohol</td><td>Any</td><td></td><td></td></tr><tr><td>Data sheets</td><td>Any</td><td></td><td></td></tr></tbody></table>

necropsy-based wild fish health assessment

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on fish populations. Adverse effects monitoring, utilizing biomarkers from the organismal to the molecular level, can be used to assess the cumulative effects on fishes and other organisms. Fish health has been used worldwide as an indicator of aquatic ecosystem health. The necropsy-based fish health assessment provides data on visible abnormalities and lesions, parasites, condition and organosomatic indices. These can be compared by site, season and sex, as well as temporally, to document change over time. Severity ratings can be assigned to various observations to calculate a fish health index for more quantitative assessment. A drawback of the necropsy-based assessment is that it is based on visual observations and condition factors, which are not as sensitive as tissue and subcellular biomarkers for sublethal effects. Additionally, it is rarely possible to identify causes or risk factors associated with observed abnormalities. So, for instance a raised lesion or "tumor" on the fins, lips or body surface may be a neoplasm. However, it could also be a response to a parasite, chronic inflammation or hyperplasia of normal cells in response to an irritant. Conversely, neoplasms, certain parasites, other infectious agents and many tissue changes are not visible and so may be underestimated. However, during the necropsy-based assessment, blood (plasma), tissues for histopathology (microscopic pathology), genomics and other molecular analyses, and otoliths for aging can be collected. These downstream analyses, together with geospatial analyses, habitat assessments, water quality and contaminant analyses can all be important in comprehensive ecosystem evaluations.

Great Lakes Areas of Concern (AOC) are geographic areas that were designated due to impairments of various beneficial uses. One of the beneficial use impairments (BUIs) at many AOC is the fish tumors or other deformities. Millions of dollars have been spent for remediation and restoration of each of these areas in order to delist the various BUIs and ultimately the AOC43. The criteria for delisting the fish tumor BUI differs from state to state (see epa.ohio.gov/portals/35/lakeerie/ohio_AOC_delisting_guidance.pdf and dnr.wi.gov/topic/GreatLakes/documents/SheboyganRiverFinalReport2008.pdf); however, as noted in the delisting documents, there is a requirement to determine the prevalence of liver tumors and in some cases skin tumors. In many cases, the prevalence is compared to a non-AOC reference site.
The fish tumor BUI was evaluated at three AOCs (St. Louis River, Milwaukee River and Sheboygan River) and a non-AOC reference site (Kewaunee River) on Lakes Superior and Michigan, utilizing a necropsy-based assessment of white sucker (Catostomus commersonii),&#160;followed by microscopic pathology of skin and liver tissue. Fish were collected from the Milwaukee, Sheboygan and Kewaunee rivers in 2012 and 201344 and from the St. Louis River in 2015 (unpublished data). Two hundred white suckers were assessed from Milwaukee, Kewaunee and St. Louis, and 193 from Sheboygan.
By definition, a tumor can be any swelling or raised area, although it is generally considered that a swelling caused by an abnormal growth of tissue with abnormal cells is either a benign or malignant neoplasm. White sucker collected from all sites exhibited a variety of external raised lesions including small, discrete white spots, larger white areas, slightly raised mucoid lesions and multilobed raised areas on the body surface and lips (Figure 6). Fish were weighed and measured to obtain a condition factor, external and internal abnormalities were documented, and skin and liver tissue was collected for histopathology.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57946/57946fig6.jpg" /> 
Figure 6: Raised skin lesions observed on white sucker from the Great Lakes.&#160;(A) A discrete white spot on the body surface. Scale bar = 5 mm. (B)&#160;A slightly raised mucoid (arrows) and multilobed lesions (a) on the posterior body surface. Scale bar = 1 cm. (C)&#160;A large, multilobed lesion on the body surface. Scale bar = 1 cm. (D)&#160;Numerous multi-lobed lesions on the lips. <a href="https://www.jove.com/files/ftp_upload/57946/57946fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The percent of fish with external tumors or raised discolored areas ranged from 15.5% at the St. Louis AOC to 58.0% at the Milwaukee AOC. In general, the discrete white spots were the least common visual lesion while the multilobed lip and body surface lesions were most common. The number of fish with observable liver nodules was low, ranging from 1.5% at Kewaunee and St. Louis to 2.5% at Milwaukee (Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Rivers and Year Sampled</td>
		</tr>
		<tr>
			<td>Visible Lesions</td>
			<td>Kewaunee 2013</td>
			<td>St. Louis 2015</td>
			<td>Sheboygan 2012</td>
			<td>Milwaukee 2013</td>
		</tr>
		<tr>
			<td>Discrete white spots</td>
			<td>16</td>
			<td>3</td>
			<td>3.1</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Mucoid</td>
			<td>20</td>
			<td>9.5</td>
			<td>9.8</td>
			<td>30.5</td>
		</tr>
		<tr>
			<td>Multilobed</td>
			<td>22.5</td>
			<td>3</td>
			<td>29.5</td>
			<td>40</td>
		</tr>
		<tr>
			<td>Total Raised Skin Abnormalitiesa</td>
			<td>46</td>
			<td>15.5</td>
			<td>38.3</td>
			<td>58</td>
		</tr>
		<tr>
			<td>Visible liver nodules</td>
			<td>1.5</td>
			<td>1.5</td>
			<td>1.6</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td colspan="5">aTotal number of fish with raised lesions. Some fish had multiple types of abnormalities.</td>
		</tr>
	</tbody>
</table>
Table 1: Necropsy-based Observations of White Sucker Collected at Great Lakes Areas of Concern and a Reference Site (Kewaunee River), Presented as a Percentage. 
Visual examination can be used to document the percent of fish with various abnormalities. However, to definitively diagnose the presence and type of neoplasia, tissues must be examined microscopically (histopathology). Upon microscopic examination, it was found that not all of the raised lesions were neoplastic. Many of the discrete white spots and the mucoid lesions, particularly at Kewaunee, were hyperplastic lesions rather than neoplasia (Table 2). Additionally, at Kewaunee and St. Louis, all of the skin tumors observed were benign papillomas. At Sheboygan and Milwaukee both papillomas and squamous cell carcinomas, malignant skin tumors, were observed (Table 2).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Rivers Sampled</td>
		</tr>
		<tr>
			<td>Neoplasm Type</td>
			<td>Kewaunee 2013</td>
			<td>St. Louis 2015</td>
			<td>Sheboygan 2012</td>
			<td>Milwaukee 2013</td>
		</tr>
		<tr>
			<td>Papilloma</td>
			<td>21</td>
			<td>5.2</td>
			<td>30.5</td>
			<td>37.5</td>
		</tr>
		<tr>
			<td>Squamous cell carcinoma</td>
			<td>0</td>
			<td>0</td>
			<td>2.1</td>
			<td>10.5</td>
		</tr>
		<tr>
			<td>Total skin neoplasms</td>
			<td>21</td>
			<td>5.2</td>
			<td>32.6</td>
			<td>48</td>
		</tr>
		<tr>
			<td>Bile duct neoplasmsa</td>
			<td>2.5</td>
			<td>4</td>
			<td>6.2</td>
			<td>9.5</td>
		</tr>
		<tr>
			<td>Hepatic cell neoplasmsb</td>
			<td>1</td>
			<td>0</td>
			<td>2.1</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Total liver neoplasms</td>
			<td>3.5</td>
			<td>4</td>
			<td>8.3</td>
			<td>15.0c</td>
		</tr>
		<tr>
			<td colspan="3">aIncludes cholangioma and cholangiocarcinoma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">bIncludes hepatic cell adenoma and hepatic cell carcinoma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">cSome fish had both bile duct and hepatic neoplasms</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Microscopically Verified Neoplastic Lesions of White Sucker Collected at Great Lakes Areas of Concern and a Reference Site (Kewaunee River), Presented as a Percentage. 
The histopathological analysis also identified liver tumors that were not identified by visual observation. While only 1.5% of the fish collected from Kewaunee and St. Louis had visible liver nodules (Table 1), 3.5% and 4.0%, respectively, had microscopically identified neoplasms (Table 2). A larger difference was seen at Sheboygan (1.6% visible versus 8.3% microscopic) and Milwaukee (2.5% visible versus 15.0% microscopic). Microscopic examination also provides a differentiation of neoplasms of bile duct versus hepatic cell origin (Table 2) and benign versus malignant tumors.

Watch this Scientific Journal Video about Necropsy-based Wild Fish Health Assessment at JoVE.com

Necropsy-based Wild Fish Health Assessment

The health of wild fishes can be used as an indicator of aquatic ecosystem health. Necropsy-based fish health assessments provide documentation of visible lesions or abnormalities, data used to calculate condition indices as well as the opportunity to collect tissues for microscopic evaluation, gene expression and other more in-depth analyses.

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on ...

necropsy-based-wild-fish-health-assessment

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Environment

17.0K Views.  U.S. Geological Survey. The health of wild fishes can be used as an indicator of aquatic ecosystem health. Necropsy-based fish health assessments provide documentation of visible lesions or abnormalities, data used to calculate condition indices as well as the opportunity to collect tissues for microscopic evaluation, gene expression and other more in-depth analyses.

Video: Necropsy-based Wild Fish Health Assessment

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological community or food web. Importantly, the size spectrum assumes that individual size, rather than species&#8217; behavioral or life history characteristics, is the primary determinant of abundance within an ecosystem. Thus, unlike traditional allometric relationships that focus on species-level data (e.g., mean species&#8217; body size vs. population density), size spectra analyses are &#8216;ataxic&#8217; &#8211; individual specimens are identified only by their size, without consideration of taxonomic identity. Size spectra models are efficient representations of traditional, complex food webs and can be used in descriptive as well as predictive contexts (e.g., predicting responses of large consumers to changes in basal resources). Empirical studies from diverse aquatic ecosystems have also reported moderate to high levels of similarity in size spectra slopes, suggesting that common processes may regulate the abundances of small and large organisms in very different settings. This is a protocol to model the community-level size spectrum in wadable streams. The protocol consists of three main steps. First, collect quantitative benthic fish and invertebrate samples that can be used to estimate local densities. Second, standardize the fish and invertebrate data by converting all individuals to ataxic units (i.e., individuals identified by size, irrespective of taxonomic identity), and summing individuals within log2 size bins. Third, use linear regression to model the relationship between ataxic M and D estimates. Detailed instructions are provided herein to complete each of these steps, including custom software to facilitate D estimation and size spectra modeling.

This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological ...

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Here, we present methods for high-throughput study of a series of the Mexican cavefish behaviors and vital staining of a mechanosensory system. These methods use free-software and custom-made scripts, providing a practical and cost-effective method for the studies of behaviors.

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among ...

Behavior

Swimming Performance Assessment in Fishes

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, pollution, hypoxia and temperature. This paper describes a flexible protocol to assess fish swimming performance using equipment in which water velocity can be controlled. The protocol involves one to several stepped increases in flow speed that are intended to cause fish to fatigue. Step speeds and their duration can be set to capture swimming abilities of different physiological and ecological relevance. Most frequently step size is set to determine critical swimming velocity (Ucrit), which is intended to capture maximum sustained swimming ability. Traditionally this test has consisted of approximately ten steps each of 20 min duration. However, steps of shorter duration (e.g. 1 min) are increasingly being utilized to capture acceleration ability or burst swimming performance. Regardless of step size, swimming tests can be repeated over time to gauge individual variation and recovery ability. Endpoints related to swimming such as measures of metabolic rate, fin use, ventilation rate, and of behavior, such as the distance between schooling fish, are often included before, during and after swimming tests. Given the diversity of fish species, the number of unexplored research questions, and the importance of many species to global ecology and economic health, studies of fish swimming performance will remain popular and invaluable for the foreseeable future.

The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour.

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, ...

Biology

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

Neuroscience

Isolation, Fixation and Characterization of Juvenile Gilthead Seabream Head Kidney Leukocytes by Flow Cytometry

Immunity is crucial for the physiological regulation of organisms, serving as the primary defense against pathogens and environmental stressors. The isolation and analysis of immune cells provide key insights into immune responses to external pressures. However, the lack of harmonized protocols for less studied species, such as marine fish, often leads to technical and analytical challenges that hamper data interpretation and a thorough understanding of species-specific immune responses. This study aimed to set up an optimized flow cytometry-based analytical procedure to characterize and determine the viability of leukocytes from the head kidney (the main hematopoietic organ in teleost fish) of juvenile gilthead seabream (Sparus aurata). The procedure began with the isolation of leukocytes through a homogenization process using Hanks&#39; balanced salt solution, followed by an optimized Percoll density gradient centrifugation method to ensure high recovery rates of leukocytes with minimal erythrocyte contamination required for efficient subsequent flow cytometry analysis. Additionally, a novel technique using a cell-reactive dye (LIVE/DEAD Fixable Dead Cell Stain Kit) was employed to distinguish viable from dead cells based on their fluorescent staining patterns. Fixation was achieved with 3.7% formaldehyde, preserving cell morphology, viability, and staining efficiency. Flow cytometry analysis successfully identified three predominant leukocyte populations: lymphocytes, monocytes, and granulocytes. This method not only allowed viability tests but also the accurate differentiation of cell types. The improvement in flow cytometry protocols represents a step forward in fish immunology by increasing the accuracy and efficiency of immune cell analysis. Furthermore, by allowing the fixation of cells for later analysis, this protocol significantly reduces the time and effort required for immune assessments, making it a valuable tool for both research and practical applications in various fields of research.

This manuscript describes the isolation and fixation of leukocytes extracted from gilthead seabream's head-kidney and the assessment of their viability by flow cytometry. This work contributes to the standardization of protocols and leverages the processing of a higher number of samples without compromising sample quality, promoting advancements in fish immunology knowledge.

Immunity is crucial for the physiological regulation of organisms, serving as the primary defense against pathogens and environmental stressors. The ...

Immunology and Infection

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump&#39;s bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

Necropsy-based Wild Fish Health Assessment

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Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Swimming Performance Assessment in Fishes

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Isolation, Fixation and Characterization of Juvenile Gilthead Seabream Head Kidney Leukocytes by Flow Cytometry

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems