Collection techniques for the analyses of pathogens in crustaceans

In the paper

“after decalcification, lobster eyes can be safely halved with a single-edged razor blade (not a scalpel) with much less damage to the delicate support network holding the ommatidia in place (Maniscalco & Shields, 2007; Magel et al. , 2009; Shields et al. , 2012a).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Maniscalco & Shields, 2007
• Partly supported

  Materials and Methods (p. 3) says the eyes were decalcified and then cut in half with a razor blade; it describes using a scalpel to remove the eyes, not to halve them. It does not specify a single-edged blade or say that this method was safer or caused less damage to the tissue supporting the ommatidia. (LLM, conf=0.88)

  cites Magel et al. , 2009 sourceExperimental — accuracy not yet measured
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Shields et al. , 2012a

In the paper

“The swimming leg, or fifth pereopod, of the blue crab, Callinectes sapidus Rathbun, 1896 has been evaluated for detecting Hematodinium infections in live juvenile crabs (Messick, 1994), but the method is not routinely used for this species and still requires a microscope for evaluation.”

What the sources say

• Supported

  The Materials and methods passage describes using the flattened fifth swimming appendage to detect Hematodinium in live, early-juvenile blue crabs with an inverted microscope. The citation is attached to that evaluation; the later statement about routine use is not attributed to Messick (1994). (LLM, conf=0.91)

  cites Messick, 1994 source

In the paper

“Neutral red is taken up differentially by phagosomes within cells, and many protozoan parasites take up the dye, whereas host hemocytes (mainly granulocytes) show only a little uptake, and stray connective tissue cells have modest uptake.”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Chatton & Poisson, 1931
• Partly supported

  The Diagnosis of Hematodinium infections section (page unavailable) says Neutral Red enters the parasite’s lysosomes while host hemocytes acquire little stain. It does not identify phagosomes, specify granulocytes as the hemocytes involved, or report modest uptake by connective-tissue cells. Its staining observation concerns particular Hematodinium infections, not protozoan parasites generally; it also notes that Hematodinium from Nephrops norvegicus does not stain. (LLM, conf=0.88)

  cites Stentiford & Shields, 2005 sourceExperimental — accuracy not yet measured

In the paper

“High-throughput sequencing has been used in some situations to help identify a pathogen but it is not practical for use in most field or fishery applications (Hewson et al. , 2014).”

What the sources say

• Partly supported

  In “Investigation of Viral Agents in SSWD-Affected Tissues” (page unavailable), the paper describes using viral metagenomic sequencing to identify a candidate sea-star pathogen. It does not assess whether high-throughput sequencing is practical for field or fishery applications, so that part of the claim is unsupported. (LLM, conf=0.86)

  cites Hewson et al. , 2014 sourceExperimental — accuracy not yet measured

In the paper

“If an assay is to be used on samples from outside the parameters used for its development, then samples and parameters should be re-validated to avoid the possibility of false negatives (Carnegie et al. , 2016).”

What the sources say

• Partly supported

  Section 3, “The paradox of advanced diagnostics” (page unavailable), says assay validation must establish that an assay performs as intended and determine which applications it suits. It does not specifically say to re-validate samples or parameters when testing outside those used in development, or link that step to avoiding false negatives. (LLM, conf=0.82)

  cites Carnegie et al. , 2016 sourceExperimental — accuracy not yet measured

In the paper

“Pathogens can for instance be present within or on a prospective host organism, but they may not necessarily be infecting that organism; hence, it is critical to confirm that the pathogen is indeed within the tissues of the host (Burreson, 2008).”

What the sources say

• Supported

  In the main text (section labeled “Published June 19”; page not provided), Burreson explains that detecting pathogen DNA does not establish infection: a parasite must overcome host barriers and become established in cells, tissues, or organs. The same section calls for confirmation by histology, which visualizes the parasite in the host. (LLM, conf=0.91)

  cites Burreson, 2008 source

In the paper

“More focused or biased collection of diseased animals is also important as it provides material for identification and documentation of the pathogen(s) and allow for the optimization of additional diagnostics (e.g., Pestal et al. , 2003; Shields et al. , 2005, 2007).”

What the sources say

• Partly supported

  The Sampling design and protocol section (page unavailable) says the researchers obtained hemolymph from every crab showing visible signs of infection, and the Sensitivity and specificity section reports confirmation of Hematodinium sp. and comparison of diagnostic preparations. This supports the paper as an example of targeted sampling for pathogen documentation and diagnostic evaluation, but it does not explicitly establish the broader claim that biased collection is important for those purposes. (LLM, conf=0.83)

  cites Pestal et al. , 2003 sourceExperimental — accuracy not yet measured
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Shields et al. , 2005, 2007

In the paper

“Freshly collected tissue samples in RNAlater® can be stored for several months at -80º C for later gene expression studies (Beale et al. , 2008).”

What the sources say

• Partly supported

  Section 2.2 reports that freshly sampled lobster tissues were stored in RNAlater, and section 2.5 describes their use for gene-expression analysis. But section 2.2 specifies −20°C, not −80°C, and gives no storage duration; it does not support the claim that samples can be kept for several months at −80°C. (LLM, conf=0.90)

  cites Beale et al. , 2008 sourceExperimental — accuracy not yet measured

In the paper

“This is crucial because many laboratories rely heavily on polymerase chain reaction (PCR) methods, which simply give presence or absence of a pathogen, and sometimes the primers are not designed or tested adequately to diagnose a pathogen correctly (Claydon et al. , 2004).”

What the sources say

• Partly supported

  The Introduction and Discussion (page not provided) describe insufficient primer specificity in the OIE WSSV PCR test and false-positive results that could lead to misdiagnosis. The paper calls the protocol an international standard, but it does not establish that many laboratories rely heavily on PCR or characterize PCR methods generally as giving only pathogen presence or absence. (LLM, conf=0.84)

  cites Claydon et al. , 2004 sourceExperimental — accuracy not yet measured

In the paper

“These dye preparations tend to form small crystals in buffer preparations and may need coarse filtration to remove them.”

In the paper

“Infections were even categorized by their relative intensity in the host.”

In the paper

“Janus green and fast green may provide some contrast to the nuclei of formalin-fixed cells with suitable results for some protozoans (JDS, pers. obs.).”

In the paper

“the hepatopancreas degrades very rapidly and is highly sensitive to weak fixation, poor buffering, and poor penetration of fixatives”

In the paper

“For small crustaceans such as copepods or small amphipods, no dissection may be necessary or small pin pricks may provide suitable entry points for fixation.”

In the paper

“In practical experience, polypropylene containers reduce adherence of cells and higher ratios of fixative can help reduce clotting, but the type of plastic container and the type of fixative should be tested prior to use (JDS, unpubl. data).”

In the paper

“Muscle and spongy connective tissues, however, can often provide useful information in post-mortem situations.”

In the paper

“Fluorochrome dyes also work well for ciliate infections, because their macro- and micronuclei can be visualized as well as dye uptake within the basal bodies of the kineties.”

In the paper

“Cells can adhere to the container used for fixation, they can clump due to handling, or the hemolymph constituents can form clots or precipitates with the fixatives, thus skewing the results or making the preparations difficult to assess.”

In the paper

“It also facilitated the later distribution of tissue samples.”

In the paper

“A significant advantage to macroscopic, or visual, diagnosis is that the method is not lethal and prevalence data can be readily incorporated into shipboard or field sampling protocols.”

In the paper

“Some specimens should, however, be fixed and preserved in 95% ethanol for later molecular identification and diagnostics.”

In the paper

“Sometimes field sampling requires fixing whole specimens. In such cases, large amphipods, small crabs (< 30 mm carapace width) and juvenile lobsters (< 30 mm carapace length) should be cut in half prior to fixation.”

In the paper

“an important feature of nearly all crustaceans is that their cuticle provides an impervious barrier to fixation; hence dissection is usually required to provide good penetration of the fixatives into the tissues of interest.”

In the paper

“For heavily sclerotized pieces, such as eyestalks (Fig. 3C), sections of carapace, claw , and whole bodies, longer decalcification is necessary (overnight or > 12 h).”

In the paper

“Many of the methods used in TEM can be difficult to employ, and tissues require tedious embedding in plastics as well as significant technical training in ultramicrotomy .”

In the paper

“Various techniques can be used to collect, identify, and monitor host populations for pathogens. These include classical methods, such as visual or histological assessment, to more refined techniques, such as simple and quantitative polymerase chain reaction assays.”

In the paper

“The ratio of tissue to fixative is critical, and it should never be less than 1:10.”

In the paper

“If fresh smears are unsuitable, prepared slides are relatively easy to make and provide a permanent record.”

In the paper

“The correct fixation of the tissues is one of the most important steps for proper diagnosis, because poor fixation can render tissues useless for histological assessment.”

In the paper

“The choice of fixative is important because tissues have different requirements for appropriate fixation and preservation.”

In the paper

“These methods can be used in the field and aboard ships, and the only significant problem is taking suitable safety precautions when transporting fixatives or using them in confined, poorly ventilated spaces.”

In the paper

“For paraffin histology , pieces of tissue no larger than 10-20 mm in size are ideal, but larger sizes can be processed depending on the nature of the tissue.”

In the paper

“Epifluorescence microscopy can be used to observe bacteria in moderate infections and special fluorochrome dyes can be used to enhance diagnoses of other pathogens as well (see below).”

In the paper

“For larger specimens, a break in the cuticle is required for the proper penetration of the fixatives; even larger specimens, such as market-sized crabs or lobsters, must be dissected and their tissues placed directly into fixative.”

In the paper

“Cuticle, foregut, or gill preparations may also require a short decalcification period for proper sectioning, whereas gonads often require different fixatives as well as longer embedding times in paraffin.”

In the paper

“Formalin is the fixative of choice for embryos because the pigments within the developing larvae are retained, whereas they are washed out with ethanol preservation.”

In the paper

“Once embedded in paraffin or plastic, specimens no longer represent a chemical hazard and can be shipped safely and easily.”

In the paper

“Lipid-rich tissues such as ovaries may require fixatives with good penetration, such as alcohol-formalin-acetic acid (AFA), longer paraffin infiltration times, or longer times in vacuum-assisted paraffin infiltration.”

In the paper

“One problem with decalcification is that the reagents, particularly formic acid, can damage the integrity of nucleic acids, and thus interfere with in situ hybridization techniques, so decalcification times should be reduced if the technique is to be used.”

In the paper

“The collection of preserved hemolymph samples can be adapted to field collections; however, there are some significant pitfalls to consider.”

In the paper

“The fixative must penetrate rapidly into the tissues to produce good results.”

In the paper

“The crustacean cuticle presents a significant barrier to the histologist.”

In the paper

“The etiology of the disease and its effect on the host population were unknown at that time (see Shields, 2013 for review).”

What the sources say

• ?Not checked — no full text of the sourcecites Shields, 2013 source

In the paper

“Three issues are absolutely critical to proper fixation: appropriate dissection, correct choice of fixative, and a suitable fixation method that includes proper ratios of tissue to fixative.”

In the paper

“The sample area should be swabbed with 70-95% ethanol if possible to avoid potential contaminants such as ciliates or diatoms that are often found on the external surfaces.”

In the paper

“SafeFix® II is easier to employ in field settings because it has less volatility .”

In the paper

“A single good fixative for both paraffin histology and TEM is problematic, but 1G:4F (1 part glutaraldehyde to 4 parts formalin; Luna, 1968) has been used with mixed results in surveys of oyster diseases (e.g., Carver et al. , 2010).”

What the sources say

• ?Not checked — no full text of the sourcecites Luna, 1968
• ?Not checked — no full text of the sourcecites Carver et al. , 2010 source

In the paper

“A minimum ratio of 1 part tissue to 10 parts preservative is best for proper preservation.”

In the paper

“Direct observation requires good photographic documentation at different magnifications for later reference; and this can be difficult aboard a ship or in rustic field conditions.”

In the paper

“Air-dried smears of crustacean hemolymph should, however, be avoided if possible, because unlike mammalian blood smears, dried hemolymph introduces too many artifacts that can make interpretation difficult, particularly when looking for cytoplasmic inclusions or nuclear alterations.”

In the paper

“The fixatives employed in TEM are hazardous, particularly glutaraldehyde and osmium tetra-oxide.”

In the paper

“Epifluorescence microscopy may also be useful for rapid diagnosis of pathogens.”

In the paper

“In most cases, the agents must have pathognomonic signs of infection, which means that the macroscopic sign of infection is specific to the pathogen causing the disease.”

In the paper

“For lightly sclerotized cuticles, such as gills and pleopods, short decalcification periods (3-6 hours) in decalcification fluid (e.g., the formic acid - sodium citrate method of Luna, 1968) may be all that is required.”

What the sources say

• ?Not checked — no full text of the sourcecites Luna, 1968

In the paper

“There are several methods for collecting hemolymph from crustaceans, and these can be adapted easily for the study of different pathogens.”

In the paper

“Storage periods should also be considered as some fixatives require refrigeration or additional post processing and cells can degrade or clump if held too long (i.e., longer than 1-2 weeks).”

In the paper

“There are two standard methods for preserving tissues for later molecular analysis: freezing specimens at -20º C or lower or preserving samples in 95% ethanol.”

In the paper

“Bouin's solution and Davidson's fixative are good general fixatives because they have good penetration into the tissues, prepare the tissue for histological stains, and give superior staining results with hematoxylin and eosin stains and other staining techniques.”

In the paper

“Direct observation of wet smears is one of the easiest methods for observing pathogens in the hemolymph, but it does require advanced training to diagnose some agents, particularly parasitic dinoflagellates, amebae, and microsporidians.”

In the paper

“Total hemocytes counts are relatively easy to quantify with a hemacytometer, but differential cell counts require training.”

In the paper

“Steedman's ester wax method is a useful alternative embedding technique for tissues rich in lipids (Humason, 1979).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Humason, 1979

In the paper

“In the case of fisheries collections, the etiological agents are typically unknown prior to an outbreak, and that can lead to poor preservation and identification of the proper agent (Shields, 2012).”

What the sources say

• ?Not checked — no full text of the sourcecites Shields, 2012 source

In the paper

“Many staining procedures can be adapted from standard histology texts (e.g., Luna 1968; Humason 1979) depending on specific pathogens and their staining attributes.”

What the sources say

• ?Not checked — no full text of the sourcecites Luna 1968
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Humason 1979

In the paper

“Histological identification and assessment of diseases requires proper fixation and handling of the tissues of interest.”

In the paper

“For work with microbial pathogens, hemolymph or other tissue samples can be adequately preserved by these methods, and the preservation can last many years.”

In the paper

“Although the method has lost its prominence in many diagnostic laboratories, it still offers a powerful means of identifying microbial pathogens such as viral and rickettsial agents.”

In the paper

“Bouin's solution, however, has two significant limitations: picric acid can be difficult to work with in certain situations, because when it dries out it can form unstable crystals that are unstable and potentially explosive, and it hydrolyzes DNA more rapidly than other fixatives, making it less desirable for molecular-based in situ hybridization techniques”

In the paper

“The etiology of a disease is often unknown prior to these outbreaks and the effect of the pathogen on the host population is poorly understood.”

• Also stated in: § INTRODUCTION · p. 2 · plain

  “In the case of fisheries collections, the etiological agents are typically unknown prior to an outbreak, and that can lead to poor preservation and identification of the proper agent (Shields, 2012).”

What the sources say

• ?Not checked — no full text of the sourcecites Shields, 2012 source

In the paper

“A data sheet was developed prior to the work that served as a checklist to ensure that tissues and data were obtained for each animal entering the study (Fig. 5).”

In the paper

“For expression or transcriptomic work, tissues should be stored routinely in fixatives such as RNAlater® (Thermo Fisher, Waltham, MA, USA) that allow gene expression studies (e.g., Hasson et al. , 1997).”

What the sources say

• ?Not checked — no full text of the sourcecites Hasson et al. , 1997 source

In the paper

“One relatively straightforward means to sample large field collections is through randomization of host individuals.”

In the paper

“The data sheet was vetted among research groups to include tissues of interest to all collaborators.”

In the paper

“We routinely scan all data sheets and retain them in a 'cloud'-based storage service with photographic documentation of histological samples and field specimens.”

In the paper

“Processed data files are stored similarly when they are completed, thus maintaining a coherent storage area for all of the data.”

In the paper

“For estimates of prevalence, it is important to have reasonable sample sizes and to minimize sample or collection biases (e.g., Gregory & Blackburn, 1991, Jovani & Tella, 2006, Shields et al. 2017).”

What the sources say

• ?Not checked — no full text of the sourcecites Gregory & Blackburn, 1991 source
• ?Not checked — no full text of the sourcecites Jovani & Tella, 2006 source
• ?Not checked — no full text of the sourcecites Shields et al. 2017 source

In the paper

“Randomization can be as simple as sampling every other host or every x th host in a trap or trawl, with the caveat that the first host in the sample stream must be randomly selected.”

In the paper

“Although this may seem trivial, it can be difficult to implement and track data sets through time, particularly after the completion of the data collection component of a study .”

In the paper

“Part of the initiative included the '100 Lobsters' project in which one laboratory served as a central point for dissecting, distributing, and archiving tissues as well as for data storage (Shields et al. , 2012a, b).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Shields et al. , 2012a, b

In the paper

“Sample data sheet from the '100 Lobsters' project used by laboratory personnel to facilitate tissue sampling, storage, shipment and receipt of samples (Shields et al. , 2012b).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Shields et al. , 2012b

In the paper

“Simple pooling is efficient when prevalence is low (< 5%), and the lowest confidence intervals can be obtained from equal pool sizes (Litvak et al. , 1994; Williams & Moffitt, 2001).”

What the sources say

• ?Not checked — no full text of the sourcecites Litvak et al. , 1994 source
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Williams & Moffitt, 2001

In the paper

“For large studies, it is often necessary to assign several identification numbers to samples because of the different data generated by collaborators.”

In the paper

“Prevalence can be biased by low sample sizes; hence, requisite sample sizes must be considered before beginning any field assessments of disease.”