- Email: [email protected]
Digestive gland inclusion bodies in queen conch (Lobatus gigas) are non-parasitic
Accepted Manuscript Digestive gland inclusion bodies in queen conch (Lobatus gigas) are non-parasitic Katie Tiley, Michelle M. Dennis, Michael R. Lewin-Smith, H. Marie Jenkins, Árni Kristmundsson, Mark A. Freeman PII: DOI: Reference:
S0022-2011(17)30505-0 https://doi.org/10.1016/j.jip.2018.07.004 YJIPA 7108
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
8 December 2017 4 July 2018 7 July 2018
Please cite this article as: Tiley, K., Dennis, M.M., Lewin-Smith, M.R., Marie Jenkins, H., Kristmundsson, A., Freeman, M.A., Digestive gland inclusion bodies in queen conch (Lobatus gigas) are non-parasitic, Journal of Invertebrate Pathology (2018), doi: https://doi.org/10.1016/j.jip.2018.07.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Digestive gland inclusion bodies in queen conch (Lobatus gigas) are non-parasitic
Katie Tileya, Michelle M. Dennisa*, Michael R. Lewin-Smithb, H. Marie Jenkinsb, Árni Kristmundssonc, Mark A. Freemana a
Center for Conservation Medicine and Ecosystem Health, Ross University School of
Veterinary Medicine, St. Kitts and Nevis b
Joint Pathology Center, Defense Health Agency, Silver Spring, Maryland, USA
c
Institute for Experimental Pathology at Keldur, University of Iceland, Reykjavík,
Iceland *Corresponding author Email addresses: MD - [email protected] KT - [email protected] MLS – [email protected] HMJ – [email protected] AK - [email protected] MF - [email protected]
1
Graphical abstract
Highlights
Large conical brown inclusion bodies in digestive gland cells are not parasites
No evidence of pathogenesis by inclusion bodies was observed
The inclusion bodies contain iron, calcium, phosphorus, sulfur, melanin, and glycoprotein
2
Abstract Unusual inclusion bodies occur within the epithelial cells of the digestive gland of queen conch, Lobatus gigas, and have previously been described as apicomplexan parasites. The aim of this study was to investigate the parasitic features of these inclusion bodies in queen conch. L. gigas from St. Kitts (Caribbean Sea) consistently (100% of n = 61) showed large numbers of ovoid to tri-bulbous dark brown inclusion bodies (15x30 µm) within vacuolar cells. Histochemical stains demonstrated iron, melanin, and glycoprotein and/or mucopolysaccharide within the inclusion bodies. Microscopic features indicative of a host response to injury were lacking in every case, as were consistent morphological forms to indicate distinct parasitic stages. Transmission electron microscopy failed to reveal cellular organelles of parasitic organisms and DNA extractions of purified inclusion bodies did not yield sufficient concentrations for successful PCR amplification. Scanning electron microscopy with energy dispersive x-ray analysis revealed a number of elements, particularly iron, within the inclusion bodies. We conclude that the inclusion bodies are not an infectious agent, and hypothesize that they represent a storage form for iron, and potentially other elements, within a protein matrix. Similar structures have been described in the digestive glands of other invertebrates, including prosobranchs.
Keywords conch, disease, histology, pathology, parasitology
3
Highlights
Large conical brown inclusion bodies in digestive gland cells are not
parasites
No evidence of pathogenesis by inclusion bodies was observed
The inclusion bodies contain iron, calcium, phosphorus, sulfur, melanin,
and glycoprotein
Abbreviations CITES - Convention on International Trade in Endangered Species HE – hematoxylin and eosin PAS – Periodic acid-Schiff TEM – transmission electron microscopy SEM/EDXA – scanning electron microscopy with energy dispersive x-ray analysis
DISCLAIMER: The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Army/Navy/Air Force, Department of Defense, or U.S. Government. The identification of specific products, scientific instrumentation, or organization is considered an integral part of the scientific endeavor and does not constitute endorsement or implied endorsement on the part of the author, DoD, or any component agency.
4
1. Introduction Queen conchs, Lobatus gigas (Linnaeus 1758) (previously Strombus gigas), are large marine gastropods and comprise one of the most significant commercial fisheries in the Caribbean Sea, including the Federation of St. Kitts and Nevis (Brownell and Stevely, 1981). L. gigas populations have collapsed in many regions due to overharvesting (Appeldoorn, 1994) and, while the species is presently listed in CITES Appendix II, stocks have not replenished despite the establishment of fishing restrictions and conservation initiatives (Avila-Poveda and Baqueiro-Cárdenas, 2009; Stoner et al., 2011). It is important to monitor L. gigas population health in order to identify impediments to local or regional recoveries, or conditions which threaten further declines. When conducting a histopathological survey of L. gigas in St. Kitts (Tiley et al., 2018), it became apparent that all individuals examined had large numbers of unusual intracytoplasmic inclusion bodies in the digestive gland epithelial cells. Identical structures have been previously described (Avila-Poveda et al., 2006), and were hypothesized to be apicomplexan parasites (Baqueiro Cárdenas et al., 2007; Gros et al., 2009). They are reportedly ubiquitous across the Caribbean region (Aldana Aranda et al., 2011), and even suspected to cause mortality or reproductive failure (Baqueiro Cárdenas et al., 2012). The aim of this study was to comprehensively assess the inclusion bodies of L. gigas digestive gland epithelial cells for features of parasites using histological, analytical chemistry, ultrastructural and molecular techniques.
2. Material and methods 2.1
Histology
5
Sixty-one L. gigas were sourced opportunistically from fishermen in cooperation with the Basseterre Fisheries Complex, harvested from the waters surrounding St. Kitts over a 13-month period (October 2015-November 2016). Animals were euthanized within hours of collection, using sedation by immersion in 3g/4L MgSO4 in seawater for 1-2 hours, followed by pithing of the ganglia posterior to the buccal mass. A 0.5 cm thick transverse section across the digestive gland was collected from a position where the coiled viscera are approximately 3 cm in width. Digestive gland tissue was either fixed in 10% neutral buffered formalin in sea water or Davidson’s solution for at least 48 hours prior to routine processing for histology. Tissues were sectioned 4 µm thick and stained with HE, Fontana Masson (with and without bleaching), Perls, Rhodanine, Von Kossa, PAS, and Alcian blue stains using standard methods (Prophet et al 1992).
2.2
Transmission Electron Microscopy
1-mm3 portions of freshly dissected digestive gland were fixed in 2.5% glutaldehyde and processed for TEM as previously described (Kristmundsson et al., 2011b). Ultrathin sections were examined using a Jeol JEM-1400 plus transmission electron microscope at the University of Iceland.
2.3
Scanning Electron Microscopy and Energy Dispersive X-ray Analysis
An unstained, 5-µm thick, de-paraffinized tissue section was mounted on a carbon disc and was examined by scanning electron microscopy with energy dispersive x-ray analysis (SEM/EDXA). SEM/EDXA was performed using a Hitachi model S3400-N scanning electron microscope (Hitachi Instruments, Inc. San Jose, CA) with an Oxford Instruments EDS energy dispersive x-
6
ray spectroscopy accessory (Oxford Instruments USA, Scotts Valley, CA). Elemental maps were obtained by using Oxford EDS Aztec software (Oxford Instruments USA, Scotts Valley, CA). The SEM/EDXA instrumentation is capable of detecting elements with atomic numbers greater than 5 (boron (B)). The SEM image was used to target structures of interest for elemental analysis by EDXA. The elemental maps were compared to the SEM image to show the distribution and colocalization of elements in a selected SEM field of interest.
2.4
DNA extraction
A suspension of inclusion bodies from three individuals, made by mechanically disrupting digestive gland tissues in saline solution, was semipurified on a Ficoll gradient by overlaying the suspension on Histopaque-1077 (Sigma), centrifuging at 2000g for 20 mins, discarding the lipid supernatant / cellular layers and collecting the pellet. The pellet was resuspended in 500 µl of saline and viewed under a compound microscope. DNA was then extracted using EurX tissue extraction kits from 100 µl of the inclusion body suspension and from ~25 mg of fresh unpurified digestive gland and stomach tissues for use as positive controls. Purified DNA concentrations were measured on a NanoDrop. PCR was performed on these extracted samples using standard 18S eukaryotic primers for control PCR (Freeman and Ogawa, 2010) and more specific primers, known to target marine gastropod/ mollusc apicomplexan parasites, in order to attempt amplification of any apicomplexan DNA (Kristmundsson et al., 2011a; Kristmundsson et al., 2015; Kristmundsson and Freeman, 2018). Marine mollusc apicomplexan DNA samples from previous studies (Pseudoklossia pectinis, Merocystis kathae, Margolisiella islandica) were used as positive controls for the apicomplexan PCRs.
7
3.
Results
3.1
Histology
Characteristic inclusion bodies were present in the digestive gland of 100% of study specimens. They were present in large numbers in the cytoplasm of digestive gland epithelial cells, specifically in vacuolated cells. These were roughly 15x30 µm, elongate to ovoid to tri-bulbous, and dark brown (Fig. 1). Occasionally they were present within the lumen of digestive gland ducts and the gastrointestinal tract. The brown pigment in the inclusion bodies stained dark brown with Fontana Masson and staining was eliminated by pre-treatment with bleach. The rims of the inclusion bodies stained pale blue with Alcian blue (pH 2.5), dark brown to black with Von Kossa, and dark pink with PAS. With Perls iron stain, small inclusions stained bright blue, and large pigmented brown inclusions were either brown or stained green-blue centrally, (Fig. 2). No inclusion bodies stained for copper with Rhodanine stain.
3.2
Microscopy
Inclusion bodies from the purified pellet suspension were observed microscopically (Fig. 4) and appeared morphologically similar to those observed on histology. The length averaged 36.34 µm (range, standard deviation: 18.7-52.5; 9.1 µm) and width averaged 17.6 µm (range, standard deviation: 9.8-27.6; 4.4 µm). Examination of tissues using transmission electron microscopy showed that the inclusion bodies consisted of electron-dense, variably compact lamellated deposits and were devoid of cellular organelles (Fig. 3). No discernable intact membranes were associated with any of the inclusion bodies.
8
3.3
DNA extraction
DNA concentrations were first visualized in an agarose gel, and also quantified using a NanoDrop. DNA extractions of the inclusion suspension did not yield sufficient DNA concentrations for PCR to be performed, with NanaoDrop values < 0.3ng/µl compared to tissue extraction values ranging between 259-419 ng/µl. PCR amplifications were successful using standard 18S primers for both stomach and digestive gland tissue extractions. However, all PCR results for apicomplexans were negative when compared to positive bands for mollusc apicomplexan DNA (Pseudoklossia pectinis, Merocystis kathae, Margolisiella islandica).
3.4
Scanning Electron Microscopy and Energy Dispersive X-ray Analysis
Brown inclusion bodies examined using SEM/EDXA contained carbon, oxygen, iron, phosphorus, sulfur, and traces of other elements including calcium. A semi-quantitative comparison of the elemental composition of small inclusions, larger inclusions and the background tissue is provided in Table 1. Figure 5 shows an SEM image and accompanying EDXA elemental maps for carbon, oxygen, iron, phosphorus and sulfur. The distribution of each of these elements within the SEM field is indicated by a color other than black. 4. Discussion Our study demonstrated characteristic inclusion bodies within the digestive gland epithelial cells of 100% of the sampled L. gigas population, consistent with other studies (Baqueiro Cárdenas et al., 2007; Gros et al., 2009). When initially described, the unusual “bottle” or conical shapes of the inclusion bodies were thought to resemble protozoal trophozoites (Baqueiro Cárdenas et al., 2007). In support of this, internal round structures were later observed with TEM and were considered to be micro- or macrogametes, or spores, suggestive of protozoal gamont and
9
sporocyst stages (Gros et al., 2009). However, in the present study and others (Aldana Aranda et al., 2011; Baqueiro Cárdenas et al., 2007), queen conch consistently had large numbers of inclusion bodies which were present year-round. It would seem highly unusual for a parasite to be 100% prevalent, and at high intensities, without any seasonal fluctuations and in multiple locations. Finally, we were unable to histologically identify associated features of the cellular injury or inflammatory response that is often observed with an infectious process, although some protozoan parasites may cause minimal tissue damage to their definitive invertebrate hosts (Kristmundsson and Freeman, 2018). Together these findings are highly unusual for and, therefore not supportive of, an infectious process. Histological examination did not show variation in forms of inclusion bodies that could be attributed to distinct protozoan stages, even when using special stains. We used TEM to better evaluate the inclusion bodies for morphologic features of parasites, but basic cellular organelles (nuclei, mitochondria) were absent within these structures. Micronemes and rhoptries, components of the apical complex that characterize certain stages of the Apicomplexa, were also lacking, nor were these structures observed in other studies examining L. gigas digestive gland epithelial cells with TEM (Gros et al., 2009; Volland et al., 2010). In addition, the inclusion bodies did not contain sufficient quantities of DNA to suggest that they are parasitic or cellular in origin. Taken together, these findings indicate that the inclusion bodies are not infectious organisms. It seems likely that the inclusion bodies represent normal physiology of L. gigas, and similar structures have been demonstrated in a variety of Strombidae species from Pacific and Atlantic regions (Volland et al., 2010).
10
‘Residual bodies’ (or ‘dense bodies’), consisting of the remnants of undigested material (Threadgold, 1976), appear histologically similar to the inclusion bodies. However, residual bodies are typically more electron dense. The presence of a number of elements in the inclusion bodies, as demonstrated by SEM/EDXA, suggests that, instead, they may have a role in element cycling, and such structures have been described in the digestive glands of other invertebrates including prosobranchs (Hyman, 1967; Voltzow, 1994). Since the inclusion bodies are shed into digestive gland ducts and expelled in the feces, as described here and by others (Baqueiro Cárdenas et al., 2007), the elements stored within the inclusion bodies are excreted to some extent. In particular, SEM/EDXA and Perl’s staining results indicate that the inclusion bodies may be important for the safe storage and/or elimination of iron. Von Kossa staining and SEM/EDXA demonstrated calcium and phosphorus within the structures as well, suggesting that they also may be involved in mineral balance. Fontana Masson stain indicated that at least some of the brown color of the inclusion bodies is the result of melanin pigment, which could account for the normal dark brown color of L. gigas digestive gland. Perhaps the inclusion bodies have a role in gut immunity of L. gigas similar to melanization in invertebrates, which is thought to facilitate encapsulation of pathogens, wound healing, and phagocytosis (Cerenius et al., 2008). Finally, the inclusion bodies staining with PAS indicated the presence of carbohydrate macromolecules such as glycoprotein or proteoglycan, and Alcian blue, indicated the presence of acidic polysaccharides such as glycosaminoglycan or mucopolysaccharide. While the present study did not quantify inclusion body abundance, Baquerio Cárdenas et al. (2012) demonstrated a negative correlation between their numbers and host gonadal maturity and spawning. Additional research is needed to better understand the function of digestive gland inclusion bodies and their role in health and reproduction.
11
5. Conclusions Digestive gland inclusion bodies in queen conch do not represent infectious organisms. They contain a number of elements, particularly iron, as well as some melanin and glycoproteins or mucopolysaccharides. We speculate that their function includes element storage and mineral processing, and additional studies are required to determine their role in health and disease.
12
Acknowledgements This work was supported by an intramural grant from Ross University School of Veterinary Medicine, Center for Conservation Medicine and Ecosystem Health. The authors thank the St. Kitts Department of Marine Resources. We also thank David Hilchie for histology and Randall Thompson, Maurice Matthew, Irene Yen, Kristi Fletcher, Hunter Burns, Tim Courtney, Michelle Farkas, and Aakansha Virwani for assistance with dissections. The authors also acknowledge the valuable contributions and expertise of Stacy Strausborger for SEM/EDXA, and of Bruce Williams for reviewing the pathology.
13
Tables Table 1: Semi-quantitative elemental composition of inclusions and background tissue by
Average
Carbon (C)
Oxygen (O)
Iron (Fe)
Phosphorus (P)
Sulfur (S)
elemental composition
Traces o Other
(n=10)
elements
<1 Background tissue
85%
13%
<1%
<1%
<1%
Ca, Mg
Inclusion bodies
71%
20%
2%
1%
3%
Ca, Mg, C Mn, Br
SEM/EDXA
14
Figure Legends
Fig.1. Digestive gland; L. gigas. Inclusion bodies (arrows) are present within the cytoplasm of vacuolated cells. Davidson’s fixative, HE stain. Bar = 100 µm.
Fig.2. Digestive gland; L. gigas. Inclusion bodies stain for iron. Davidson’s fixative, Perls stain. Bar = 50 µm.
Fig.3. Digestive gland; L. gigas. Inclusion bodies are variably electron dense and somewhat laminar (arrows). TEM. Bar = 10 µm.
Fig.4. Purified digestive gland inclusion bodies; L. gigas. Inclusion bodies are ovoid to tribulbous, dark brown, and internal structures cannot be discerned. Unstained. Bar = 20 µm.
Fig.5. SEM-EDXA. SEM image of L. gigas digestive gland showing intracytoplasmic inclusion bodies (upper). EDXA elemental maps for carbon (C), oxygen (O), iron (Fe), phosphorus (P) and sulfur (S) (lower).
15
References Aldana Aranda, D., Frenkiel, L., Brulé, T., Montero, J., Baqueiro Cárdenas, E., 2011. Occurrence of Apicomplexa-like structures in the digestive gland of Strombus gigas throughout the Caribbean. J Invertebr Pathol. 106, 174-8. Avila-Poveda, O.H., Aldana-Aranda, D., Baqueiro-Cárdenas, E.R., 2006. Histology of selected regions of the alimentary system of Strombus gigas Linnaeus, 1758 (Caenogastropoda: Strombidae). Amer Malac Bull. 21, 93-98. Avila-Poveda, O. H., Baqueiro-Cárdenas, E. R., 2009. Reproductive cycle of Strombus gigas Linnaeus 1758 (Caenogastropoda: Strombidae) from Archipelago of San Andres, Providencia and Santa Catalina, Colombia. Invertebr Repr Dev. 53, 1-12. Appeldoorn, R. S., Queen Conch Management and Research: Status, Needs and Priorities. . In: R. S. Appeldoorn, B. Rodriguez, Eds.), Queen Conch Biology, Fisheries and Mariculture. Caracas: Fundacion Cientifica Los Roques, 1994, pp. 301-319. Baqueiro Cárdenas, E., Frenkiel, L., Zetina Zarate, A., Aldana Aranda, D., 2007. Coccidian (Apicomplexa) parasite infecting Strombus gigas Linné, 1758 digestive gland. J Shellfish Res. 26, 319-321. Baqueiro Cárdenas, E., Montero, J., Frenkiel, L., Aldana Aranda, D., 2012. Attenuated reproduction of Strombus gigas by an Apicomplexa: Emeriidae-like parasite in the digestive gland. J Invertebr Pathol. 110, 398-400. Brownell, W. N., Stevely, J. M., 1981. The biology, fisheries and management of the queen conch, Strombus gigas. Marine Fisheries Review. 43, 1-12. Cerenius, L., Lee, B. L., & Sӧderhӓll, K, 2008. The proPO-system: pros and cons for its role in invertebrate immunity. Trends Immunol. 29, 263-71.
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Freeman, M. A., Ogawa, K., 2010. Variation in the small subunit ribosomal DNA confirms that Udonella (Monogenea: Udonellidae) is a species-rich group. Int J Parasitol. 40, 255-264. Gros, O., Frenkiel, L., Aldana Aranda, D., 2009. Structural analysis of the digestive gland of the queen conch Strombus gigas Linnaeus, 1758 and its intracellular parasites. J Molluscan Stud. 75, 59-68. Hyman, L. H., VIII. Class Gastropoda: Subclass Prosobranchia. In: L. H. Hyman, (Ed.), The Invertebrates: Mollusca I (Volume VI). McGraw-Hill Book Company, 1967. Kristmundsson, Á., Erlingsdóttir, Á., Freeman, M. A., 2015. Is an apicomplexan parasite responsible for the collapse of the Iceland scallop (Chlamys islandica) stock? PLoS ONE 10(12). Kristmundsson, Á., Freeman, M. A., 2018. Harmless sea snail parasite causes mass mortalities in numerous commercial scallop populations in the northern hemisphere. Sci. Rep. (accepted: in press). Kristmundsson, Á., Helgason, S., Bambir, S. H., Eydal, M., Freeman, M. A., 2011a. Margolisiella islandica sp. nov. (Apicomplexa: Eimeridae) infecting Iceland scallop Chlamys islandica (Muller, 1776) in Icelandic waters. J Invertebr Pathol. 108, 139-46. Kristmundsson, Á., Helgason, S., Bambir, S. H., Eydal, M., Freeman, M. A., 2011b. Previously unknown apicomplexan species infecting Iceland scallop, Chlamys islandica (Muller, 1776), queen scallop, Aequipecten opercularis L., and king scallop, Pecten maximus L. J Invertebr Pathol. 108, 147-55. Prophet, E.B., B. Mills, J.B. Arrington, and L.H. Sobin (Editors). 1992. Armed Forces Institute of Pathology - Laboratory Methods in Histotechnology. American Registry of Pathology, AFIP, Washington, DC. 279 p.
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Stoner, A., Davis, M., Booker, C., Evidence for a Significant Decline in Queen Conch in the Bahamas, Including a Population in a Marine Protected Area. Gulf and Caribbean Fisheries Institute, Vol. 64, 2011, pp. 349-361. Threadgold, L. T., The Ultrastructure of the Cytosome. II. The Hyaloplasm, Endoplasmic Reticulum, and Cell Inclusions. The Ultrastructure of the Animal Cell, 1976, pp. 215294. Tiley, K., Freeman, M. A., Dennis, M. M., 2018. Pathology and reproductive health of queen conch (Lobatus gigas) in St. Kitts. J Invertebr Pathol. In Press. Volland, J. M., Frenkiel, L., Aldana Aranda, D., Gros, O., 2010. Occurrence of Sporozoa-like microorganisms in the digestive gland of various species of Strombidae. J Molluscan Stud. 76, 196-198. Voltzow, J., Gastropoda: Prosobranchia. In: F. W. Harrison, A. J. Kohn, (Eds.), Microscopic Anatomy of Invertebrates: Volume 5: Mollusca One: Monoplacophora, Aplacophora, Polyplacophora and Gastropoda. Wiley-Blackwell, 1994, pp. 111-252.
18
S0022-2011(17)30505-0 https://doi.org/10.1016/j.jip.2018.07.004 YJIPA 7108
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
8 December 2017 4 July 2018 7 July 2018
Please cite this article as: Tiley, K., Dennis, M.M., Lewin-Smith, M.R., Marie Jenkins, H., Kristmundsson, A., Freeman, M.A., Digestive gland inclusion bodies in queen conch (Lobatus gigas) are non-parasitic, Journal of Invertebrate Pathology (2018), doi: https://doi.org/10.1016/j.jip.2018.07.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Digestive gland inclusion bodies in queen conch (Lobatus gigas) are non-parasitic
Katie Tileya, Michelle M. Dennisa*, Michael R. Lewin-Smithb, H. Marie Jenkinsb, Árni Kristmundssonc, Mark A. Freemana a
Center for Conservation Medicine and Ecosystem Health, Ross University School of
Veterinary Medicine, St. Kitts and Nevis b
Joint Pathology Center, Defense Health Agency, Silver Spring, Maryland, USA
c
Institute for Experimental Pathology at Keldur, University of Iceland, Reykjavík,
Iceland *Corresponding author Email addresses: MD - [email protected] KT - [email protected] MLS – [email protected] HMJ – [email protected] AK - [email protected] MF - [email protected]
1
Graphical abstract
Highlights
Large conical brown inclusion bodies in digestive gland cells are not parasites
No evidence of pathogenesis by inclusion bodies was observed
The inclusion bodies contain iron, calcium, phosphorus, sulfur, melanin, and glycoprotein
2
Abstract Unusual inclusion bodies occur within the epithelial cells of the digestive gland of queen conch, Lobatus gigas, and have previously been described as apicomplexan parasites. The aim of this study was to investigate the parasitic features of these inclusion bodies in queen conch. L. gigas from St. Kitts (Caribbean Sea) consistently (100% of n = 61) showed large numbers of ovoid to tri-bulbous dark brown inclusion bodies (15x30 µm) within vacuolar cells. Histochemical stains demonstrated iron, melanin, and glycoprotein and/or mucopolysaccharide within the inclusion bodies. Microscopic features indicative of a host response to injury were lacking in every case, as were consistent morphological forms to indicate distinct parasitic stages. Transmission electron microscopy failed to reveal cellular organelles of parasitic organisms and DNA extractions of purified inclusion bodies did not yield sufficient concentrations for successful PCR amplification. Scanning electron microscopy with energy dispersive x-ray analysis revealed a number of elements, particularly iron, within the inclusion bodies. We conclude that the inclusion bodies are not an infectious agent, and hypothesize that they represent a storage form for iron, and potentially other elements, within a protein matrix. Similar structures have been described in the digestive glands of other invertebrates, including prosobranchs.
Keywords conch, disease, histology, pathology, parasitology
3
Highlights
Large conical brown inclusion bodies in digestive gland cells are not
parasites
No evidence of pathogenesis by inclusion bodies was observed
The inclusion bodies contain iron, calcium, phosphorus, sulfur, melanin,
and glycoprotein
Abbreviations CITES - Convention on International Trade in Endangered Species HE – hematoxylin and eosin PAS – Periodic acid-Schiff TEM – transmission electron microscopy SEM/EDXA – scanning electron microscopy with energy dispersive x-ray analysis
DISCLAIMER: The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Army/Navy/Air Force, Department of Defense, or U.S. Government. The identification of specific products, scientific instrumentation, or organization is considered an integral part of the scientific endeavor and does not constitute endorsement or implied endorsement on the part of the author, DoD, or any component agency.
4
1. Introduction Queen conchs, Lobatus gigas (Linnaeus 1758) (previously Strombus gigas), are large marine gastropods and comprise one of the most significant commercial fisheries in the Caribbean Sea, including the Federation of St. Kitts and Nevis (Brownell and Stevely, 1981). L. gigas populations have collapsed in many regions due to overharvesting (Appeldoorn, 1994) and, while the species is presently listed in CITES Appendix II, stocks have not replenished despite the establishment of fishing restrictions and conservation initiatives (Avila-Poveda and Baqueiro-Cárdenas, 2009; Stoner et al., 2011). It is important to monitor L. gigas population health in order to identify impediments to local or regional recoveries, or conditions which threaten further declines. When conducting a histopathological survey of L. gigas in St. Kitts (Tiley et al., 2018), it became apparent that all individuals examined had large numbers of unusual intracytoplasmic inclusion bodies in the digestive gland epithelial cells. Identical structures have been previously described (Avila-Poveda et al., 2006), and were hypothesized to be apicomplexan parasites (Baqueiro Cárdenas et al., 2007; Gros et al., 2009). They are reportedly ubiquitous across the Caribbean region (Aldana Aranda et al., 2011), and even suspected to cause mortality or reproductive failure (Baqueiro Cárdenas et al., 2012). The aim of this study was to comprehensively assess the inclusion bodies of L. gigas digestive gland epithelial cells for features of parasites using histological, analytical chemistry, ultrastructural and molecular techniques.
2. Material and methods 2.1
Histology
5
Sixty-one L. gigas were sourced opportunistically from fishermen in cooperation with the Basseterre Fisheries Complex, harvested from the waters surrounding St. Kitts over a 13-month period (October 2015-November 2016). Animals were euthanized within hours of collection, using sedation by immersion in 3g/4L MgSO4 in seawater for 1-2 hours, followed by pithing of the ganglia posterior to the buccal mass. A 0.5 cm thick transverse section across the digestive gland was collected from a position where the coiled viscera are approximately 3 cm in width. Digestive gland tissue was either fixed in 10% neutral buffered formalin in sea water or Davidson’s solution for at least 48 hours prior to routine processing for histology. Tissues were sectioned 4 µm thick and stained with HE, Fontana Masson (with and without bleaching), Perls, Rhodanine, Von Kossa, PAS, and Alcian blue stains using standard methods (Prophet et al 1992).
2.2
Transmission Electron Microscopy
1-mm3 portions of freshly dissected digestive gland were fixed in 2.5% glutaldehyde and processed for TEM as previously described (Kristmundsson et al., 2011b). Ultrathin sections were examined using a Jeol JEM-1400 plus transmission electron microscope at the University of Iceland.
2.3
Scanning Electron Microscopy and Energy Dispersive X-ray Analysis
An unstained, 5-µm thick, de-paraffinized tissue section was mounted on a carbon disc and was examined by scanning electron microscopy with energy dispersive x-ray analysis (SEM/EDXA). SEM/EDXA was performed using a Hitachi model S3400-N scanning electron microscope (Hitachi Instruments, Inc. San Jose, CA) with an Oxford Instruments EDS energy dispersive x-
6
ray spectroscopy accessory (Oxford Instruments USA, Scotts Valley, CA). Elemental maps were obtained by using Oxford EDS Aztec software (Oxford Instruments USA, Scotts Valley, CA). The SEM/EDXA instrumentation is capable of detecting elements with atomic numbers greater than 5 (boron (B)). The SEM image was used to target structures of interest for elemental analysis by EDXA. The elemental maps were compared to the SEM image to show the distribution and colocalization of elements in a selected SEM field of interest.
2.4
DNA extraction
A suspension of inclusion bodies from three individuals, made by mechanically disrupting digestive gland tissues in saline solution, was semipurified on a Ficoll gradient by overlaying the suspension on Histopaque-1077 (Sigma), centrifuging at 2000g for 20 mins, discarding the lipid supernatant / cellular layers and collecting the pellet. The pellet was resuspended in 500 µl of saline and viewed under a compound microscope. DNA was then extracted using EurX tissue extraction kits from 100 µl of the inclusion body suspension and from ~25 mg of fresh unpurified digestive gland and stomach tissues for use as positive controls. Purified DNA concentrations were measured on a NanoDrop. PCR was performed on these extracted samples using standard 18S eukaryotic primers for control PCR (Freeman and Ogawa, 2010) and more specific primers, known to target marine gastropod/ mollusc apicomplexan parasites, in order to attempt amplification of any apicomplexan DNA (Kristmundsson et al., 2011a; Kristmundsson et al., 2015; Kristmundsson and Freeman, 2018). Marine mollusc apicomplexan DNA samples from previous studies (Pseudoklossia pectinis, Merocystis kathae, Margolisiella islandica) were used as positive controls for the apicomplexan PCRs.
7
3.
Results
3.1
Histology
Characteristic inclusion bodies were present in the digestive gland of 100% of study specimens. They were present in large numbers in the cytoplasm of digestive gland epithelial cells, specifically in vacuolated cells. These were roughly 15x30 µm, elongate to ovoid to tri-bulbous, and dark brown (Fig. 1). Occasionally they were present within the lumen of digestive gland ducts and the gastrointestinal tract. The brown pigment in the inclusion bodies stained dark brown with Fontana Masson and staining was eliminated by pre-treatment with bleach. The rims of the inclusion bodies stained pale blue with Alcian blue (pH 2.5), dark brown to black with Von Kossa, and dark pink with PAS. With Perls iron stain, small inclusions stained bright blue, and large pigmented brown inclusions were either brown or stained green-blue centrally, (Fig. 2). No inclusion bodies stained for copper with Rhodanine stain.
3.2
Microscopy
Inclusion bodies from the purified pellet suspension were observed microscopically (Fig. 4) and appeared morphologically similar to those observed on histology. The length averaged 36.34 µm (range, standard deviation: 18.7-52.5; 9.1 µm) and width averaged 17.6 µm (range, standard deviation: 9.8-27.6; 4.4 µm). Examination of tissues using transmission electron microscopy showed that the inclusion bodies consisted of electron-dense, variably compact lamellated deposits and were devoid of cellular organelles (Fig. 3). No discernable intact membranes were associated with any of the inclusion bodies.
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3.3
DNA extraction
DNA concentrations were first visualized in an agarose gel, and also quantified using a NanoDrop. DNA extractions of the inclusion suspension did not yield sufficient DNA concentrations for PCR to be performed, with NanaoDrop values < 0.3ng/µl compared to tissue extraction values ranging between 259-419 ng/µl. PCR amplifications were successful using standard 18S primers for both stomach and digestive gland tissue extractions. However, all PCR results for apicomplexans were negative when compared to positive bands for mollusc apicomplexan DNA (Pseudoklossia pectinis, Merocystis kathae, Margolisiella islandica).
3.4
Scanning Electron Microscopy and Energy Dispersive X-ray Analysis
Brown inclusion bodies examined using SEM/EDXA contained carbon, oxygen, iron, phosphorus, sulfur, and traces of other elements including calcium. A semi-quantitative comparison of the elemental composition of small inclusions, larger inclusions and the background tissue is provided in Table 1. Figure 5 shows an SEM image and accompanying EDXA elemental maps for carbon, oxygen, iron, phosphorus and sulfur. The distribution of each of these elements within the SEM field is indicated by a color other than black. 4. Discussion Our study demonstrated characteristic inclusion bodies within the digestive gland epithelial cells of 100% of the sampled L. gigas population, consistent with other studies (Baqueiro Cárdenas et al., 2007; Gros et al., 2009). When initially described, the unusual “bottle” or conical shapes of the inclusion bodies were thought to resemble protozoal trophozoites (Baqueiro Cárdenas et al., 2007). In support of this, internal round structures were later observed with TEM and were considered to be micro- or macrogametes, or spores, suggestive of protozoal gamont and
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sporocyst stages (Gros et al., 2009). However, in the present study and others (Aldana Aranda et al., 2011; Baqueiro Cárdenas et al., 2007), queen conch consistently had large numbers of inclusion bodies which were present year-round. It would seem highly unusual for a parasite to be 100% prevalent, and at high intensities, without any seasonal fluctuations and in multiple locations. Finally, we were unable to histologically identify associated features of the cellular injury or inflammatory response that is often observed with an infectious process, although some protozoan parasites may cause minimal tissue damage to their definitive invertebrate hosts (Kristmundsson and Freeman, 2018). Together these findings are highly unusual for and, therefore not supportive of, an infectious process. Histological examination did not show variation in forms of inclusion bodies that could be attributed to distinct protozoan stages, even when using special stains. We used TEM to better evaluate the inclusion bodies for morphologic features of parasites, but basic cellular organelles (nuclei, mitochondria) were absent within these structures. Micronemes and rhoptries, components of the apical complex that characterize certain stages of the Apicomplexa, were also lacking, nor were these structures observed in other studies examining L. gigas digestive gland epithelial cells with TEM (Gros et al., 2009; Volland et al., 2010). In addition, the inclusion bodies did not contain sufficient quantities of DNA to suggest that they are parasitic or cellular in origin. Taken together, these findings indicate that the inclusion bodies are not infectious organisms. It seems likely that the inclusion bodies represent normal physiology of L. gigas, and similar structures have been demonstrated in a variety of Strombidae species from Pacific and Atlantic regions (Volland et al., 2010).
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‘Residual bodies’ (or ‘dense bodies’), consisting of the remnants of undigested material (Threadgold, 1976), appear histologically similar to the inclusion bodies. However, residual bodies are typically more electron dense. The presence of a number of elements in the inclusion bodies, as demonstrated by SEM/EDXA, suggests that, instead, they may have a role in element cycling, and such structures have been described in the digestive glands of other invertebrates including prosobranchs (Hyman, 1967; Voltzow, 1994). Since the inclusion bodies are shed into digestive gland ducts and expelled in the feces, as described here and by others (Baqueiro Cárdenas et al., 2007), the elements stored within the inclusion bodies are excreted to some extent. In particular, SEM/EDXA and Perl’s staining results indicate that the inclusion bodies may be important for the safe storage and/or elimination of iron. Von Kossa staining and SEM/EDXA demonstrated calcium and phosphorus within the structures as well, suggesting that they also may be involved in mineral balance. Fontana Masson stain indicated that at least some of the brown color of the inclusion bodies is the result of melanin pigment, which could account for the normal dark brown color of L. gigas digestive gland. Perhaps the inclusion bodies have a role in gut immunity of L. gigas similar to melanization in invertebrates, which is thought to facilitate encapsulation of pathogens, wound healing, and phagocytosis (Cerenius et al., 2008). Finally, the inclusion bodies staining with PAS indicated the presence of carbohydrate macromolecules such as glycoprotein or proteoglycan, and Alcian blue, indicated the presence of acidic polysaccharides such as glycosaminoglycan or mucopolysaccharide. While the present study did not quantify inclusion body abundance, Baquerio Cárdenas et al. (2012) demonstrated a negative correlation between their numbers and host gonadal maturity and spawning. Additional research is needed to better understand the function of digestive gland inclusion bodies and their role in health and reproduction.
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5. Conclusions Digestive gland inclusion bodies in queen conch do not represent infectious organisms. They contain a number of elements, particularly iron, as well as some melanin and glycoproteins or mucopolysaccharides. We speculate that their function includes element storage and mineral processing, and additional studies are required to determine their role in health and disease.
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Acknowledgements This work was supported by an intramural grant from Ross University School of Veterinary Medicine, Center for Conservation Medicine and Ecosystem Health. The authors thank the St. Kitts Department of Marine Resources. We also thank David Hilchie for histology and Randall Thompson, Maurice Matthew, Irene Yen, Kristi Fletcher, Hunter Burns, Tim Courtney, Michelle Farkas, and Aakansha Virwani for assistance with dissections. The authors also acknowledge the valuable contributions and expertise of Stacy Strausborger for SEM/EDXA, and of Bruce Williams for reviewing the pathology.
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Tables Table 1: Semi-quantitative elemental composition of inclusions and background tissue by
Average
Carbon (C)
Oxygen (O)
Iron (Fe)
Phosphorus (P)
Sulfur (S)
elemental composition
Traces o Other
(n=10)
elements
<1 Background tissue
85%
13%
<1%
<1%
<1%
Ca, Mg
Inclusion bodies
71%
20%
2%
1%
3%
Ca, Mg, C Mn, Br
SEM/EDXA
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Figure Legends
Fig.1. Digestive gland; L. gigas. Inclusion bodies (arrows) are present within the cytoplasm of vacuolated cells. Davidson’s fixative, HE stain. Bar = 100 µm.
Fig.2. Digestive gland; L. gigas. Inclusion bodies stain for iron. Davidson’s fixative, Perls stain. Bar = 50 µm.
Fig.3. Digestive gland; L. gigas. Inclusion bodies are variably electron dense and somewhat laminar (arrows). TEM. Bar = 10 µm.
Fig.4. Purified digestive gland inclusion bodies; L. gigas. Inclusion bodies are ovoid to tribulbous, dark brown, and internal structures cannot be discerned. Unstained. Bar = 20 µm.
Fig.5. SEM-EDXA. SEM image of L. gigas digestive gland showing intracytoplasmic inclusion bodies (upper). EDXA elemental maps for carbon (C), oxygen (O), iron (Fe), phosphorus (P) and sulfur (S) (lower).
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