Morphological features of mermithid nematodes (Mermithidae) were studied with scanning electron microscopy, using hexamethyldisilazane-air drying in comparison with critical point drying via liquid carbon dioxide. Although general morphologic preservation of both HMDS-dried and CP-dried specimens was similar, structural features of the complex cuticle and internal organization were more easily resolved at higher magnifications in the HMDS-dried nematodes. These features include the superficial cuticular annulations, the fibrillar inner cuticle and peg-like microtrabeculae. The previously undescribed microtrabeculae are of special interest since they may facilitate an interaction of the mermithid (and perhaps nameatodes in general) musculature with its body wall that, at least in part, may account for the unique thrashing locomotion so characteristic of these organisms

Bowen, William R.

Good, Barry H.

Hemann, Christopher C.

Johnson, Arthur A.

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 44 Article 61990Mermithid Nematodes: SEM ObservationsComparing Hexamethyldisilazane and CriticalPoint Drying MethodsWilliam R. BowenUniversity of Arkansas at Little RockChristopher C. HemannHendrix CollegeArthur A. JohnsonHendrix CollegeBarry H. GoodUniversity of Arkansas at Little RockFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Biochemistry CommonsThis article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able toread, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking priorpermission from the publisher or the author.This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academyof Science by an authorized editor of ScholarWorks@UARK. For more information, please contact ccmiddle@uark.edu, drowens@uark.edu,scholar@uark.edu.Recommended CitationBowen, William R.; Hemann, Christopher C.; Johnson, Arthur A.; and Good, Barry H. (1990) "Mermithid Nematodes: SEMObservations Comparing Hexamethyldisilazane and Critical Point Drying Methods," Journal of the Arkansas Academy of Science: Vol.44 , Article 6.Available at: http://scholarworks.uark.edu/jaas/vol44/iss1/6WILLIAMR. BOWEN1 CHRISTOPHER C. HEMANNDepartment of Biology ARTHUR A. JOHNSONUniversity of Arkansas at Little Rock Department of BiologyLittle Rock, AR 72204 Hendrix CollegeDMIDepaiUniversity of >cklithidnematodes (Mermithidae) wereluucie ana iminnuiauons, me Tioniiar inner cuncie ana peg-iiKe microiraDecuiae. me previously uiiubbuiiubulicrotrabeculae are of special interest since they may facilitate an interaction of the mermithid (and perhapslematodes in general) musculature with its body wall that, at least inpart, may account for the uniquehrashing locomotion so characteristic of these organisms.iiiuaruior routine s]uiierent lecnniques or preparing pianiparasitic nemaioues ior acivi.rhese techniques included different fixatives in combination with dif-erent transitional fluids (liquid carbon dioxide or freon) forCP or freezeIrying. He found that, for most nematode genera, glutaraldehyde fix-ition and freeze drying provided an adequate preservation ofnematodenorphology. Nation (1983), however, described another approch topecimen drying in which fixed dehydrated insects were immersed intexamethyldisilazane (HMDS) and air dried. HMDS drying not onlyiroved to be a satisfactory substitute for carbon dioxide critical pointIrying for insects, but bacteria as well (Giammara, et al., 1987). HMDSIrying significantly decreases the time required for specimen process-tig. The purpose of this study was toevaluate the applicability ofHMDSIrying to nematodes for SEM examination.MATERIALS AND METHODSAdult chironomids, host to parasitic imagocidal mermithid.Dry. isuiaieu inaic auu icmaic s>p<iian uniaentiiiea genus ana oinyaromermis, ana aauiis oij^anceimer-nis, were fixed in cacodylate buffered 2°7o glutaraldehyde for 4 days,tostfixed in 1% osmium tetroxide for 30 min., and dehydrated in eithern ethanol or acetone series. Acetone-dehydrated specimens were criticalloint dried using a Samdri 780A drier and liquidcarbon dioxide as theransitional fluid. Alcohol-dehydrated specimens were immersed inIMDSfor 10 min. and substantially air dried. Some specimens werePresent address: Department of Biology, Jacksonville State Univer-ity, Jacksonville, AL 36265RESULTS AND DISCUSSION:OMPARISON OF HMDS- ANDCP-DRIED MERMITHIDSnaioues except inaiinternal simuiiiai morpnoiogicai ieaiures as cepnaiic papiuae anu muuui vrig.lapiuae ana anai pore were aiso eviaeni in mermniiiu nuues.nboth females and males, the superficial cuticular annulations (SCA)o clearly evident in transmission electron micrographs (TEM) of thelody region (Batson, 1979; Poinar, 1983) are barely discernible in SEMnicrographs of CP-dried specimens (Fig. 1). Cross-sectional views ofmermithids broken in two were badly fragmented and pro-ided little information about the internal organization of theselematodes. Resolution of most structures was limited at magnificationsbove 4.000X.Morphologic preservation of HMDS-dried mermithids was similaro that of CP-dried specimens except that cuticular detail and internal•reservation were significantly better at higher magnifications. Althoughhe head and body region of a HMDS-dried mermithid female in Fig.exhibits some shrinkage, the specimen clearly shows the fine SCAs..ne &i_as exmou a parauel oanaing pauern inme ooay regions ings.:, 3, 6). The pattern of SCAs inone mermithid species was distinctlyegular (Figs. 2, 3), whereas in another itwas highly irregular (Fig. 6).'hese initialobservations suggest that SEM analysis of SCA patternsnay have some taxonomic value for mermithid nematodes. Inthe headegion, however, the pattern of SCAs becomes reoriented around themphids so the SCAs now occur at right angles to those found in theiody regions (Fig. 4) and form a circular pattern at the most anteriornd (Fig. 2). Cross-sectional views ofbroken HMDS-dried mermithids/ere remarkably similar to that expected with freeze-fracture techni-lues. Fig. 5. shows the nematode trophosome withitsnumerous lipopro-16Journal of the Arkansas Academy of Science, Vol. 44 [1990], Art. 6Published by Arkansas Academy of Science, 1990Mgure3. view 01 ooay 01 MMUS-aneanyaromerma, uiuwmg wcu-ucuucu irupnu^umc wun numcro(arrow). 786X.rigure 6. Cuticle of HMDS-dried Hydromermis, showing exposed subcuticular fibers (arrow) and irregular SCADrn^nnHlnnc Arlraneac A/^aHomu r\§ Qf-ion^ UaI yl/1 -IQOA17Journal of the Arkansas Academy of Science, Vol. 44 [1990], Art. 6http://scholarworks.uark.edu/jaas/vol44/iss1/6Proceedings Arkansas Academy of Science, Vol. 44, 199018Mermithid Nematodes: SEM Observations Comparing Hexamethyldisilazane and Critical Point Drying Methodstein globules as ifithad been sections (Fig. 5). The unique muscle bandsof the nematode were not distinct in this view. Torn cuticular surfacesof HMDS-dried mermithids (Figs. 6, 8), however, clearly demonstratedthe 3-dimensional aspects of the inner fibrillar zone of the mermithidcuticle previously described byBatson (1979) from TEMmicrographs.SEveral layers of fibers were evident, the individual fibers of which lieparallel one to another. All fiber layers together constituted an innerfiber belt oriented diagonally to the SCAs of the cuticle surface. Sincethe SCAs were perpendicular to the longitudinal axis of the nematodebody, this fibrous belt is also oriented diagonally along the entire lengthof the nematode body.Although general morphologic preservation ofboth HMDS-dried andCPD-dried specimens was similar, itis apparent that resolving the struc-tural features of the complex cuticle and internal organization ofmermithid nematodes with the scanning electron microscopy are bestachieved with HMDS-dried specimens.MICROTRABECULAEBeneath the hypodermis, an elaborate system ofminute peg-like struc-tures covering the outer surface of the muscle bands were discoveredinHMDS-dried mermithids (Figs. 7, 8). Since the presence of these struc-tures has not been previously reported in the nematodes, including themermithids (Batson, 1979; Poinar, 1983), wehave termed these struc-tures microtrabeculae.These microtrabeculae project from the muscle surface toward thehypodermis ofthe body wall.They are arranged inparallel series perpen-dicular to the body wall and the longitudinal muscle bands (Figs. 7,8). They appear to physically interact with the hypodermis, as suggestedby tissue, presumably remnants of the hypodermis, that remains at-tached to some microtrabeculae (Fig. 8).The functional role of the microtrabeculae is interesting and remainsto be elucidated. The microtrabeculae however do appear to structual-ly interact withthe hypodermis (and therefore the cuticle and body wall).This could mean that they provide a structural means in which the mer-mithid's longitudinal musculature and body wall is integrated into asingle biomechanical unit. Ifso, the microtrabeculae may then assumea major, previously unrecognized, role in the biomechanics of thethrashing locomotion so characteristic of the mermithids and perhapsnematodes in genera. Study of these structures and their relationshipis being continued.LITERATURE CITEDBATSON, B.S. 1979. Body wall of juvenile and adult Gastromermisboophlhorae (Nematoda: Ultrastructure and nutritional role. Int.J. Parasitol. 9:495-503.EISENBACK, J.D. 1986. A comparison of techniques useful forpreparing nematodes for scanning electron microscopy. J. Nematol.18:479-487.GIAMMARA,B.W. DeVRIES, R. BAKER, J. DOBBINS, and J.HANKEL.1987. Hexamethyldisilazane drying for rapid detectionof bacteria in implact specimens. Proc. Electron Microsc. Soc.Amer. 45:878-879.NATION, J.L. 1983. A new method using hexamethyldisilazanefor preparation of soft insect tissues for scanning electronmicroscopy. Stain Technol. 58:347-351.POINAR, CO., JR. 1975. Entomogenous nematodes. E.J. Brill,Leiden. 317 pp.POINAR, CO., JR. 1983. The natural history of nematodes. Prentice-Hall, Englewood Cliffs (NJ). 323 pp.POSTEK, M.T., K.S. HOWARD, A.H. JOHNSON, and K.L.McMICHAEL.1980. Scanning electron microscopy: a studenthandbook. Ladd Research Industries, Burlington (VT).305 pp.Figure 7. Torn outer body region of HMDS-dried Hydromermis,showing exposed fiber layers (0 of the cuticle and thenumerous microtrabeculae (t) arranged inparallel series onthe outer surface of a muscle band. 16.620X.Figure 8. Same as Fig. 7, showing hypodermal remnants attached tomicrotrabeculae exposed beneath the cuticle. 11,400X.18Journal of the Arkansas Academy of Science, Vol. 44 [1990], Art. 6Published by Arkansas Academy of Science, 1990

Mermithid Nematodes: SEM Observations Comparing Hexamethyldisilazane and Critical Point Drying Methods

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 44 Article 6 1990 Mermithid Nematodes: SEM Observations Comparing Hexamethyldisilazane and Critical Point Drying Methods William R. Bowen University of Arkansas at Little Rock Christopher C. Hemann Hendrix College Arthur A. Johnson Hendrix College Barry H. Good University of Arkansas at Little Rock Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Biochemistry Commons Recommended Citation Bowen, William R.; Hemann, Christopher C.; Johnson, Arthur A.; and Good, Barry H. (1990) "Mermithid Nematodes: SEM Observations Comparing Hexamethyldisilazane and Critical Point Drying Methods," Journal of the Arkansas Academy of Science: Vol. 44 , Article 6. Available at: https://scholarworks.uark.edu/jaas/vol44/iss1/6 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu. WILLIAMR. BOWEN1 CHRISTOPHER C. HEMANNDepartment of Biology ARTHUR A. JOHNSONUniversity of Arkansas at Little Rock Department of BiologyLittle Rock, AR 72204 Hendrix CollegeDMIDepaiUniversity of >cklithidnematodes (Mermithidae) wereluucie ana iminnuiauons, me Tioniiar inner cuncie ana peg-iiKe microiraDecuiae. me previously uiiubbuiiubulicrotrabeculae are of special interest since they may facilitate an interaction of the mermithid (and perhapslematodes in general) musculature with its body wall that, at least inpart, may account for the uniquehrashing locomotion so characteristic of these organisms.iiiuaruior routine s]uiierent lecnniques or preparing pianiparasitic nemaioues ior acivi.rhese techniques included different fixatives in combination with dif-erent transitional fluids (liquid carbon dioxide or freon) forCP or freezeIrying. He found that, for most nematode genera, glutaraldehyde fix-ition and freeze drying provided an adequate preservation ofnematodenorphology. Nation (1983), however, described another approch topecimen drying in which fixed dehydrated insects were immersed intexamethyldisilazane (HMDS) and air dried. HMDS drying not onlyiroved to be a satisfactory substitute for carbon dioxide critical pointIrying for insects, but bacteria as well (Giammara, et al., 1987). HMDSIrying significantly decreases the time required for specimen process-tig. The purpose of this study was toevaluate the applicability ofHMDSIrying to nematodes for SEM examination.MATERIALS AND METHODSAdult chironomids, host to parasitic imagocidal mermithid.Dry. isuiaieu inaic auu icmaic s>p<iian uniaentiiiea genus ana oinyaromermis, ana aauiis oij^anceimer-nis, were fixed in cacodylate buffered 2°7o glutaraldehyde for 4 days,tostfixed in 1% osmium tetroxide for 30 min., and dehydrated in eithern ethanol or acetone series. Acetone-dehydrated specimens were criticalloint dried using a Samdri 780A drier and liquidcarbon dioxide as theransitional fluid. Alcohol-dehydrated specimens were immersed inIMDSfor 10 min. and substantially air dried. Some specimens werePresent address: Department of Biology, Jacksonville State Univer-ity, Jacksonville, AL 36265RESULTS AND DISCUSSION:OMPARISON OF HMDS- ANDCP-DRIED MERMITHIDSnaioues except inaiinternal simuiiiai morpnoiogicai ieaiures as cepnaiic papiuae anu muuui vrig.lapiuae ana anai pore were aiso eviaeni in mermniiiu nuues.nboth females and males, the superficial cuticular annulations (SCA)o clearly evident in transmission electron micrographs (TEM) of thelody region (Batson, 1979; Poinar, 1983) are barely discernible in SEMnicrographs of CP-dried specimens (Fig. 1). Cross-sectional views ofmermithids broken in two were badly fragmented and pro-ided little information about the internal organization of theselematodes. Resolution of most structures was limited at magnificationsbove 4.000X.Morphologic preservation of HMDS-dried mermithids was similaro that of CP-dried specimens except that cuticular detail and internal•reservation were significantly better at higher magnifications. Althoughhe head and body region of a HMDS-dried mermithid female in Fig.exhibits some shrinkage, the specimen clearly shows the fine SCAs..ne &i_as exmou a parauel oanaing pauern inme ooay regions ings.:, 3, 6). The pattern of SCAs inone mermithid species was distinctlyegular (Figs. 2, 3), whereas in another itwas highly irregular (Fig. 6).'hese initialobservations suggest that SEM analysis of SCA patternsnay have some taxonomic value for mermithid nematodes. Inthe headegion, however, the pattern of SCAs becomes reoriented around themphids so the SCAs now occur at right angles to those found in theiody regions (Fig. 4) and form a circular pattern at the most anteriornd (Fig. 2). Cross-sectional views ofbroken HMDS-dried mermithids/ere remarkably similar to that expected with freeze-fracture techni-lues. Fig. 5. shows the nematode trophosome withitsnumerous lipopro-16Journal of the Arkansas Academy of Science, Vol. 44 [1990], Art. 6Published by Arkansas Academy of Science, 1990Mgure3. view 01 ooay 01 MMUS-aneanyaromerma, uiuwmg wcu-ucuucu irupnu^umc wun numcro(arrow). 786X.rigure 6. Cuticle of HMDS-dried Hydromermis, showing exposed subcuticular fibers (arrow) and irregular SCADrn^nnHlnnc Arlraneac A/^aHomu r\§ Qf-ion^ UaI yl/1 -IQOA17Journal of the Arkansas Academy of Science, Vol. 44 [1990], Art. 6https://scholarworks.uark.edu/jaas/vol44/iss1/6Proceedings Arkansas Academy of Science, Vol. 44, 199018Mermithid Nematodes: SEM Observations Comparing Hexamethyldisilazane and Critical Point Drying Methodstein globules as ifithad been sections (Fig. 5). The unique muscle bandsof the nematode were not distinct in this view. Torn cuticular surfacesof HMDS-dried mermithids (Figs. 6, 8), however, clearly demonstratedthe 3-dimensional aspects of the inner fibrillar zone of the mermithidcuticle previously described byBatson (1979) from TEMmicrographs.SEveral layers of fibers were evident, the individual fibers of which lieparallel one to another. All fiber layers together constituted an innerfiber belt oriented diagonally to the SCAs of the cuticle surface. Sincethe SCAs were perpendicular to the longitudinal axis of the nematodebody, this fibrous belt is also oriented diagonally along the entire lengthof the nematode body.Although general morphologic preservation ofboth HMDS-dried andCPD-dried specimens was similar, itis apparent that resolving the struc-tural features of the complex cuticle and internal organization ofmermithid nematodes with the scanning electron microscopy are bestachieved with HMDS-dried specimens.MICROTRABECULAEBeneath the hypodermis, an elaborate system ofminute peg-like struc-tures covering the outer surface of the muscle bands were discoveredinHMDS-dried mermithids (Figs. 7, 8). Since the presence of these struc-tures has not been previously reported in the nematodes, including themermithids (Batson, 1979; Poinar, 1983), wehave termed these struc-tures microtrabeculae.These microtrabeculae project from the muscle surface toward thehypodermis ofthe body wall.They are arranged inparallel series perpen-dicular to the body wall and the longitudinal muscle bands (Figs. 7,8). They appear to physically interact with the hypodermis, as suggestedby tissue, presumably remnants of the hypodermis, that remains at-tached to some microtrabeculae (Fig. 8).The functional role of the microtrabeculae is interesting and remainsto be elucidated. The microtrabeculae however do appear to structual-ly interact withthe hypodermis (and therefore the cuticle and body wall).This could mean that they provide a structural means in which the mer-mithid's longitudinal musculature and body wall is integrated into asingle biomechanical unit. Ifso, the microtrabeculae may then assumea major, previously unrecognized, role in the biomechanics of thethrashing locomotion so characteristic of the mermithids and perhapsnematodes in genera. Study of these structures and their relationshipis being continued.LITERATURE CITEDBATSON, B.S. 1979. Body wall of juvenile and adult Gastromermisboophlhorae (Nematoda: Ultrastructure and nutritional role. Int.J. Parasitol. 9:495-503.EISENBACK, J.D. 1986. A comparison of techniques useful forpreparing nematodes for scanning electron microscopy. J. Nematol.18:479-487.GIAMMARA,B.W. DeVRIES, R. BAKER, J. DOBBINS, and J.HANKEL.1987. Hexamethyldisilazane drying for rapid detectionof bacteria in implact specimens. Proc. Electron Microsc. Soc.Amer. 45:878-879.NATION, J.L. 1983. A new method using hexamethyldisilazanefor preparation of soft insect tissues for scanning electronmicroscopy. Stain Technol. 58:347-351.POINAR, CO., JR. 1975. Entomogenous nematodes. E.J. Brill,Leiden. 317 pp.POINAR, CO., JR. 1983. The natural history of nematodes. Prentice-Hall, Englewood Cliffs (NJ). 323 pp.POSTEK, M.T., K.S. HOWARD, A.H. JOHNSON, and K.L.McMICHAEL.1980. Scanning electron microscopy: a studenthandbook. Ladd Research Industries, Burlington (VT).305 pp.Figure 7. Torn outer body region of HMDS-dried Hydromermis,showing exposed fiber layers (0 of the cuticle and thenumerous microtrabeculae (t) arranged inparallel series onthe outer surface of a muscle band. 16.620X.Figure 8. Same as Fig. 7, showing hypodermal remnants attached tomicrotrabeculae exposed beneath the cuticle. 11,400X.18Journal of the Arkansas Academy of Science, Vol. 44 [1990], Art. 6Published by Arkansas Academy of Science, 1990

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Mermithid Nematodes: SEM Observations Comparing Hexamethyldisilazane and Critical Point Drying Methods

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