Survey of slug-parasitic nematodes in East and West Flanders, Belgium and description of Angiostoma gandavensis n. sp. (Nematoda: Angiostomidae) from arionid slugs.

A survey for slug-associated nematodes in five locations of East and West Flanders in Belgium revealed the presence of one new and six known slug-parasitic nematodes, Agfa flexilis (Dujardin, 1845), Alloionema appendiculatum (Schneider, 1859), Angiostoma dentiferum (Mengert, 1953), Angiostoma limacis (Dujardin, 1845), Angiostoma norvegicum (Ross et al., 2017) and Phasmarhabditis hermaphrodita (Schneider, 1859). Angiostoma norvegicum and P. hermaphrodita are recorded for the first time in Belgium. The six known species are documented by light microscopy (LM) microphotographs and informative DNA sequences. Angiostoma gandavensis n. sp. (Angiostomatidae), discovered from arionid slugs, is described based on light microscopy, scanning electron microscopy (SEM) and molecular data. Based on analyses of D2D3 expansion segment of 28S and 18S rDNA sequences, this new species is found to be related to A. limacis, A. norvegicum, A. margaretae (Ross et al., 2011) and A. milacis (Ivanova and Wilson, 2009). The new species can be distinguished from these others based on morphological characters such as the distinctive mucronate structures at the tail tip of both sexes, presence of lateral ala, reflexed female ovaries and the number and arrangement pattern of male genital papillae.

First Record of Trichodorus primitivus and Morphological and Molecular Identification of Longidorus elongatus from Canada.

During July 2008, sandy loam soil samples were collected near the rhizosphere of Kentucky bluegrass (Poa pratensis L.) and tall fescue (Festuca arundinacea Schreb.) at a depth of 15 to 20 cm from Caledonia Park, Toronto, Canada. Samples were suspended in water and nematodes were collected on sieves with 250- and 74-µm openings. Among the nematodes recovered were one species of Longidorus Micoletzky, 1922 (4) and one species of Trichodorus Cobb, 1913. The Trichodorus species (2), was identified as T. primitivus (de Man, 1880) Micoletzky, 1922, in which females have rod-like sclerotized pieces parallel to the vagina lumen, each with dimensions of 3.1 × 1.2 µm and at a distance of 2 to 3 µm from each other; and males have spicules with a wide nonoffset capitulum and very narrow blade, three ventromedian cervical papillae (CP), and three precloacal supplements with the anterior one at the level of the capitulum of retracted spicules. Males showed unusual variation in the position of CP2 (i.e., posterior to the onchiostyle region, except for one specimen with CP2 located within the posterior onchiostyle region, which is typical for the species). The morphometric data for nine males are: L, 665 to 805 µm; a, 23.0 to 27.5; b, 3.7 to 5.2; c, 46 to 56; onchiostyle, 47 to 52 µm; and spicules, 32.5 to 40.5 µm. Measurements for 13 females are: L, 630 to 775 µm; a, 20.5 to 24.8; b, 3.5 to 5.5; c, 75.5 to 158.5; and V, 54 to 61%. To our knowledge, this is the first report of T. primitivus in Canada. According to Chen et al. (1), the Longidorus species was identified as L. elongatus (de Man, 1876) Micoletzky, 1922 (3). The morphometric data for females (n = 13) are: L, 5.1 to 6.0 mm; a, 78.5 to 106.5; b, 11.5 to 13.5; c, 86.0 to 120.5; V, 45.5 to 53.0%; odontostyle, 79 to 91 µm; odontophore, 57.5 to 66.5 µm; and tail length, 44.5 to 59.0 µm. Males were not found. Four juvenile stages were identified. J1 (n = 2) with a body length of 1.1, 1.2 mm and replacement/functional odontostyle 59, 59/53.5, 54.5 µm, J2 (n = 2) with L: 1.7, 2.0 mm and replacement/functional odontostyle 62.5, 68.0/58.7 µm, 59.0, J3 (n = 2): L: 2.8, 3.0 mm and replacement/functional odontostyle 76.5, 77/66.5, 67.0 µm and J4 (n = 2) with L: 3.6, 3.8 mm and replacement/functional odontostyle 87.0, 90.5/75.0, 77.5 µm. Due to large morphometric overlap in Longidorus species identification, 2,472 bp of the near full-length 18S and the internal transcribed spacer 1 region of rDNA (Accession No. GU199044) were sequenced. The Blastn search of the partial 18S revealed 100% identity with a population of L. elongatus from Scotland (GenBank No. AY687992, 1,707 bp compared), 99% identity (3 bp difference, 1,707 bp compared) with a population of L. elongatus from Iran (EU503141) and 99% identity (4 bp difference, 1,707 bp compared) with a population of L. elongatus (AF036594, sample location unknown). A Blastn search of the 18S and ITS region revealed only 1 to 3 bp differences with two populations of L. elongatus from Switzerland (AJ549986 and AJ549987) and a population of L. elongatus (AF511417) from Scotland. These molecular data further confirmed the identity of the population from Canada to be L. elongatus. References: (1) Q. Chen et al. Fundam. Appl. Nematol. 20:15, 1997. (2) W. Decraemer. Kluwer Academic Publishers, Dordrecht, the Netherlands, 1995. (3) J. G. de Man. Tijdschr. Ned. Dierk. Ver. 2:78, 1876. (4) H. Micoletzky. Archiv. Naturgesch. 87:1, 1922.

A new approach to identify species in Xiphinema americanum sensu lato.

Four putative species belonging to the X. americanum group are known to transmit American nepoviruses and these nematodes and viruses are listed in European quarantine legislation. Identification of species in this group is therefore of particular importance for phytosanitary purposes, but is problematic because of the similar morphology of the putative species. As part of the Synthesys project BE-TAF 1769, eight collaborative institutions contributed material to Fera for study. Video technology allowed the best records of type material and in conjunction with photographic images created a 'virtual' collection of images that relies less on the deteriorating quality of museum specimens. Revised definitions of lip region and tail shape are considered to be the most stable characters for differentiation. Position of the amphidial aperture, development of the odontostyle collar and some established morphometric characters are considered of limited use although they retain value for latter stages of identification.

First Report of Trichodorus variopapillatus (Nematoda: Trichodoridae) from the Czech Republic.

Members of the Trichodoridae can cause substantial crop losses directly by feeding on plant roots and indirectly as vectors of tobraviruses; both vector and virus are polyphagous. In April of 2008, soil samples from the rhizosphere of Ulmus minor Mill in a deciduous broadleaf forest at Krivoklat yielded a population of Trichodorus variopapillatus Hooper, 1972. Nematodes were identified by morphological and morphometric characters as well as by molecular analysis. For classical identification, specimens were extracted from soil by a decanting-sieving method, heat killed and fixed in triethanolamine formalin, and processed and mounted in anhydrous glycerin. For molecular analysis, specimens were stored at -20°C in 1 M NaCl. Specimens largely agreed with T. variopapillatus (1,3). Average morphometric data of five male specimens are: body length 766 µm; body width 33 µm; onchiostyle length 55 µm; and spicule length 43 µm. Number of anterior ventromedian cervical papillae and number of precloacal supplements was three each. Spicules are regularly curved and the manubrium is knob-like. Morphometric data of two female specimens are: body length 663 and 858 µm; body width 29 and 38 µm; onchiostyle length 52 and 53 µm; V 54 and 57%. Refractive thickenings at the vulva are very large and quandrangular in shape in the lateral optical section. Identification of these nematodes was further verified by sequencing two regions of rDNA (18S gene and D2/D3 expansion segments of the 28S gene). Single female and male specimens from NaCl storage were transferred to 0.5-ml Eppendorf tubes containing 0.25M NaOH. Total genomic DNA was prepared by a rapid technique (4). The 18S gene was amplified in three fragments using the primer SSU_F_04 + SSU_R_09 (first fragment), SSU_F_22 + SSU_R_13 (second fragment), and SSU_F_23 + SSU_R_81 (third fragment). D2/D3 expansion segments of the large subunit of rDNA were amplified using the forward primer D2A and the reverse primer D3B (2). The regions were sequenced in both directions after purification of PCR products. The sequences of female and male specimens were identical. The sequences were deposited in GenBank with Accession Nos. GQ148719 (28S) and GQ148719 (18S). The length of 18S was 1,760 bp and D2/D3 was 786 bp. The obtained sequences were compared by BLAST in NCBI. The D2/D3 sequence is not available in GenBank for T. variopapillatus. The best BLAST hits were obtained with Trichodorus species. BLAST results of 18S sequence showed 5% divergence (76 substitutions) after trimming unequal ends with published sequence of T. variopapillatus Accession No. AY284841. All substitutions were confirmed from the chromatographs. To our knowledge, this is the first report of T. variopapillatus associated with U. minor in the Czech Republic. References: (1) W. Decraemer and P. Baujard. Fundam. Appl. Nematol. 21:37 1998. (2) P. De Ley et al. Nematology 1:591, 1999. (3) D. Hooper. Nematologica 17:59, 1972. (4) J. M. Stanton. Australas. Plant Pathol. 27:112, 1998.

Simultaneous measurements of ossicular velocity and intracochlear pressure leading to the cochlear input impedance in gerbil.

Recent measurements of three-dimensional stapes motion in gerbil indicated that the piston component of stapes motion was the primary contributor to intracochlear pressure. In order to make a detailed correlation between stapes piston motion and intracochlear pressure behind the stapes, simultaneous pressure and motion measurements were undertaken. We found that the scala vestibuli pressure followed the piston component of the stapes velocity with high fidelity, reinforcing our previous finding that the piston motion of the stapes was the main stimulus to the cochlea. The present data allowed us to calculate cochlear input impedance and power flow into the cochlea. Both the amplitude and phase of the impedance were quite flat with frequency from 3 kHz to at least 30 kHz, with a phase that was primarily resistive. With constant stimulus pressure in the ear canal the intracochlear pressure at the stapes has been previously shown to be approximately flat with frequency through a wide range, and coupling that result with the present findings indicates that the power that flows into the cochlea is quite flat from about 3 to 30 kHz. The observed wide-band intracochlear pressure and power flow are consistent with the wide-band audiogram of the gerbil.

First Report of the Dagger Nematode Xiphinema dentatum (Nematoda: Longidoridae) in a Deciduous Forest in the Czech Republic.

Xiphinema species are migratory ectoparasitic nematodes that feed on an extensive range of hosts and several species are vectors of nepoviruses. These long nematodes are readily distinguished from most other plant parasitic nematodes by a long stylet with forked odontostyle and flanged odontophore. In May of 2005, a sample from the rhizosphere of Carpinus betulus and Acer platanoides in a forest near Silnicna, South Moravia yielded a population of Xiphinema dentatum Sturhan, 1978. X. dentatum previously has been reported to be associated with several forest and grassland species in Germany, the former Yugoslavia, and Slovakia. Specimens were extracted from soil by decanting-sieving. A few female specimens were stored at -20°C in 1 M NaCl, and the rest of the specimens were heat killed, fixed in triethanolamine formalin, and mounted in anhydrous glycerin. In 2007, nematodes from permanent slides were identified by morphological and morphometrical characters (3): female body C shaped in fixed specimens, lip region offset by a depression, reproductive system amphidelphic with the presence of well developed pseudo Z-organ, and tail broadly convex-conoid to regularly hemispherical; main average morphometric of females were body length 3.6 mm, total stylet length 220 µm, vulva position 46%, and tail ratio 0.66. Identification of these nematodes was further verified by sequencing cytochrome oxidase subunit 1 (cox1) of mtDNA and D2/D3 expansion segments of large subunit rDNA. Two individual female specimens from NaCl storage were transferred to 0.5-ml Eppendorf tubes containing 0.25 M NaOH. Total genomic DNA was prepared by a rapid technique (4). The cox1 gene was amplified using forward primer COIF (5'-GAT TTT TTG GKC ATC CWG ARG-3') and reverse primer COIR (5'-CWA CAT AAT AAG TAT CAT G-3') (2). D2/D3 expansion segments of large subunit of rDNA were amplified using forward primer D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG -3') and reverse primer D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') (1). The regions were sequenced in both directions after purification of PCR products from gel slices with a Qiagen gel extraction kit (Qiagen, Hilden, Germany). The sequences of two individual females were identical. The sequences were deposited in GenBank (Accession Nos. EU781537 [cox1] and EU781538 [D2/D3]). The length of cox1 was 393 bp and D2/D3 was 786 bp. The obtained sequences were compared by BLAST in NCBI. The cox1 gene sequence is not available in GenBank for X. dentatum, but the best BLAST hits were logically obtained with Xiphinema species. BLAST results of D2/D3 sequence showed strong similarities (99.6%) with X. dentatum Accession No. AY601627 and only a three nucleotide difference was observed in the beginning of the 5' end. To our knowledge, this is the first report of X. dentatum associated with deciduous forest trees in the Czech Republic. Forests are the main terrestrial ecosystems and rich in species diversity and are of great importance as natural resources. Therefore, information on these plant parasitic nematodes from forests would be useful because they are a component of the continental forest diversity. References: (1) P. De Ley et al. Nematology 2:591, 1999. (2) Y. He et al. J. Mol. Evol. 61:819, 2005. (3) P. A. A. Loof and M. Luc. Syst. Parasitol. 16:35, 1990. (4) J. M. Stanton. Australas. Plant Pathol. 27:112, 1998.

First Report of Trichodorus similis from the Czech Republic (Nematoda: Trichodoridae).

Members of the Trichodoridae can cause substantial crop losses directly by feeding on plant roots and indirectly as vectors of tobraviruses; both vector and virus are polyphagous. Although trichodorid nematodes are important pests of agricultural crops, no data are available on the presence or extent of these nematodes in the Czech Republic. In June 2005, three soil samples from the rhizosphere of a Quercus sp. at Cerveny Vrch yielded a population of Trichodorus similis Seinhorst, 1963. Specimens were extracted from soil by a decanting-sieving method, heat killed, and fixed in triethanolamine formalin (TAF), and processed and mounted in anhydrous glycerin. Nematodes were identified by morphological and morphometrical characters (2). Classical identification of these nematodes was further verified by molecular study. A single, male specimen was temporarily mounted in distilled water on a glass slide and relaxed with gentle heat. Measurements and photographs were taken, and the specimen was transferred to a 0.5-ml Eppendorf tube containing 0.25 M NaOH. Total genomic DNA was prepared by a rapid technique (4). Morphometric data of the male specimen used for DNA study are: body length 867 µm; body width 81 µm; onchiostyle length 44 µm; spicule length 36 µm; distance of anterior cervical papilla (CP)1 from anterior end 39 µm, CP1 to CP2 25 µm, CP2 to CP3 22 µm; posterior precloacal supplement (SP1) to cloacal opening 27 µm, distance SP1 to SP2 32 µm, distance SP2 to SP3 39 µm. The following primers were used in the PCR reaction: species-specific sense primer SIMIREV2 (5'-CACTCGTCGGACTCAAACC-3') and universal antisense primer UNIVERSAL (5'-CCCGTCGCTACTACCGATT-3') (1). A single fragment of approximately 452 bp was amplified. The D2 and D3 expansion regions of the large subunit 28S rDNA were amplified using the primer D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B (5'-TCGGAAGGAACCAGCTACTA-3' (3). The region was sequenced after purification of PCR products from the gel slice with a Qiagen gel purification kit (Qiagen Inc., Valencia, CA). The obtained sequence was deposited in Genbank (Accession No. DQ832183). The obtained sequence was compared by BLAST in NCBI and the results showed strong similarities with T. similis (Accession No. AM180730). To our knowledge, this is the first report of T. similis associated with a deciduous forest in the Czech Republic. Taking into account the agricultural importance of trichodorids and tobraviruses as plant pathogens, there is a need for a comprehensive survey of these taxa in the Czech Republic. The damage level threshold is in the case of virus vector species equivalent to a single nematode. Therefore, information on these plant parasites would be useful for developing nematode management strategies. References: (1) K. Boutsika et al. Plant Pathol. 53:110, 2004. (2) W. Decraemer and P. Baujard. Fundam. Appl. Nematol. 21:37, 1998. (3) P. De Ley et al. Nematology 2:591, 1999. (4) J. M. Stanton. Australas. Plant Pathol. 27:112, 1998.

Thickness distribution of fresh eardrums of cat obtained with confocal microscopy.

The aim of this study was to measure the spatial thickness distribution of the cat tympanic membrane (TM), a very thin, virtually transparent and delicate biological membrane. Axial fluorescence images taken perpendicular through isolated TM were recorded for five different cats using confocal laser scanning microscopy. Thickness was measured on the cross-section of the membranes in the axial images. A correction for focal shift due to refractive-index mismatch was applied. Similar thickness distributions were obtained in all measured samples (n = 9). The pars tensa had a rather constant thickness in the central region between the annulus and manubrium. The thickness increased steeply toward the peripheral rim. Thickness was smallest in the inferior region, with values ranging between 5.5 and 9 microm in the central part and up to 50 microm near the annulus. More superiorly, thickness was slightly higher, up to 20 microm, between the annulus and manubrium. The anterior part was thicker than the posterior side. These findings are strongly different from a current value in the literature. Our data allow a more precise representation of the eardrum in mathematical models, which are a prerequisite for a better understanding of middle-ear mechanics. The optical sectioning technique of the confocal microscope did not result in any preparation artifacts and was therefore also used to quantify shrinkage due to preparation of histological sections of TMs.

First Report of the Stubby-Root Nematode Paratrichodorus teres Associated with Artichoke (Cynara scolymus) in Greece.

The polyphagous stubby-root nematode species, Paratrichodorus teres (Hooper) Siddiqi, was first described from soil under lettuce near Norwich, UK and subsequently reported from South Africa and the United States, but predominantly from temperate regions within Europe (4). P. teres is one of 13 economically important trichodorid species known to be vectors of Tobacco rattle virus (TRV) (4). Artichokes planted during 2000 in a field located in the Kandia area of the Argolis Region, Greece (37°32'N, 22°56'E) exhibited symptoms of a virus infection. Sampling was done to ascertain the presence of Longidorus fasciatus, a vector of artichoke Italian latent nepovirus known to occur in the area (1,4). In addition to L. fasciatus, an unknown trichodorid species and Tylenchorhyncus sp. were recovered from the root zone of artichoke at a number of sites within the field. Measurements and morphological examination of the female (n = 13, body length = 741.7 ± 25.5 µm, onchiostyle = 43.7 ± 0.8 µm, and position of vulva from anterior region relative to total body length V% = 53.8 ± 0.4 µm) and male (n =1, body length = 720.5 µm, onchiostyle = 43.5 µm, spicule length = 51.7 µm, and number of ventromedian precloacal supplements = 3) trichodorids isolated from soil samples conformed to the original description of P. teres and the generic polytomous key (2). Furthermore, morphological identification was supported by molecular data. DNA was extracted from seven individual trichodorids, each of which were placed into separate 0.5-ml micro-centrifuge tubes containing 20 µl of 0.25 M NaOH and incubated at 25°C overnight. Thereafter, samples were incubated at 99°C for 3 min and 10 µl of 0.25 M HCl, 5 µl of 0.5 M Tris-HCl, (pH 8.0) and 5 µl of 2% Triton X-100 were added to each tube. Samples were incubated at 99°C for a further 3 min and stored at -20°C. Template DNA was amplified using polymerase chain reaction with primers specific for 18S rDNA and sequenced (3). The resultant consensus sequence had 99.8% homology to P. teres populations isolated from Portugal and good homology (95 to 98%) with five other Paratrichodorus spp. listed on public sequence databases, e.g., NCBI GenBank. This constitutes a new geographic record and a possible association of P. teres on artichoke. References: (1) D. J. F. Brown et al. Eur. J. Plant Pathol. 103:501, 1997. (2) W. Decraemer. Fundam. Appl. Nematol. 21:37, 1998. (3) C. M. G. Oliveira et al. J. Nematol. 36:153, 2004. (4) C. E. Taylor and D. J. F. Brown. Nematode Vectors of Plant Viruses, CAB International Mycological Institute, Wallingford, UK, 1997.

Three-dimensional modelling of the middle-ear ossicular chain using a commercial high-resolution X-ray CT scanner.

The quantitative measurement of the three-dimensional (3-D) anatomy of the ear is of great importance in the making of teaching models and the design of mathematical models of parts of the ear, and also for the interpretation and presentation of experimental results. This article describes how we used virtual sections from a commercial high-resolution X-ray computed tomography (CT) scanner to make realistic 3-D anatomical models for various applications in our middle-ear research. The important problem of registration of the 3-D model within the experimental reference frame is discussed. The commercial X-ray CT apparatus is also compared with X-ray CT using synchrotron radiation, with magnetic resonance microscopy, with fluorescence optical sectioning, and with physical (histological) serial sections.

Elastic properties of human aortas in relation to age and atherosclerosis: a structural model.

A new structural model is described for the tension-radius relationship of blood vessels, taking into account their mechanically important constituents: collagen, elastin and smooth muscle. The model has four characteristic parameters: EC, the Young's modulus of the collagen fibres; ESE, the Young's modulus of the combined smooth-muscle/elastin network; epsilon mu, the amount of strain at which the high stiffness region on the tension-radius curve is reached, and eta an indicator for the degree of collagen fibre stretching. The structural stiffness of the wall constituents is reflected by EC and ESE whereas the global stiffness of the entire blood vessel is described by epsilon mu and eta. All these elasticity parameters are pressure independent, in contrast to generally quoted values for the incremental modulus or vascular compliance which are strongly pressure dependent. Hence, an objective comparison of the mechanical properties for various types of blood vessel, based on the present model parameters, has been made possible. The model was successfully fitted to tension-radius data of 65 human aortas, age range 30-88 years, with moderate or severe atherosclerosis. The structural as well as the global stiffness changes with age, e.g. collagen stiffness shows a ninefold increase over 60 years. Global stiffness depends on atherosclerosis.

Area change and volume displacement of the human tympanic membrane under static pressure.

Direct measurements are presented of the area change and volume displacement of a human tympanic membrane under static pressures in the range of -1.6 KPa to +1.6 kPa. The area change is given separately for the pars tensa and the pars flaccida. For the pars tensa a strong asymmetry in area change under positive and negative pressure is observed. The volume displacement is also given separately for the pars tensa and the pars flaccida. The volume displacement of the entire TM agrees very well with volume displacement data in literature on tympanometry. It is shown further that a linear relationship between umbo displacement and volume displacement exists. The compliance of the tympanic membrane under static pressure load is compared to acoustic compliance measurements at low frequencies, and found to be a factor 2.5 higher than the compliance at 500 Hz.

Human tympanic membrane deformation under static pressure.

The effect of static pressures in the range of plus and minus 1.6 kPa on the shape of tympanic membrane is measured using a non-contacting optical technique on a fresh human temporal bone. Full field data of the deformation are presented as well as cross-sections along two major directions. Strong asymmetry between medial and lateral movements is demonstrated. The displacement of the umbo is compared to other work. The rotation angle of the manubrium in function of pressure is calculated and also compared to other work. It is demonstrated that the rotation angels can not account for the measured movement of the umbo, which leads to the conclusion that for static high pressure levels the classical hypothesis of rotation around a fixed axis has to be abandoned. The comparison with data of TM displacement under dynamic stimuli is discussed.

Modelling the malleus vibration as a rigid body motion with one rotational and one translational degree of freedom.

Vibration of a set of points distributed along the manubrium of cat was measured with a heterodyne interferometer in response to sinusoidal acoustic signals. The observed motion did not fit pure rotation of the malleus around a fixed axis coinciding with the anterior mallar and posterior incudal ligament as is classically assumed. As a first approximation a model of motion consisting of a rotational and a translational component was used. At low frequencies the rotation is mostly predominant, but the situation may be entirely reversed at mid and high frequencies. The presence of a translation besides rotation was also found at some frequencies in the motion of the human malleus.

Effect of middle ear components on eardrum quasi-static deformation.

Eardrum deformation induced by quasi-static middle ear pressure was studied at progressive stages of dissection of gerbil temporal bones. With our high resolution moiré interferometer we recorded the shape and deformation of the eardrum along a line perpendicular to the manubrium and through the umbo, at different middle ear pressures. The deformation was measured from the medial side, after serially removing the cochlea, removing the stapes, cutting the tensor tympani, exposing the incudo-mallear joint, and cutting the anterior bony process which connects the malleus to the tympanic bone. The mean displacement as a function of pressure was also determined at all stages of dissection. Removing the cochlea and stapes, and cutting tensor tympani has no effect on static eardrum deformation. Exposing the incudo-mallear joint increases eardrum movement, and cutting the anterior bony connection between malleus and temporal bone strongly changes eardrum rest position and further increases its displacement.

Heterodyne interferometer measurements of the frequency response of the manubrium tip in cat.

A heterodyne interferometer proved to be a very accurate tool to measure amplitude and phase of the malleus response during acoustical stimulation. It was shown that to obtain equal accuracy in the acoustical pressure measurements, pressure response must be remeasured at short time intervals. At frequencies above 4 kHz various gross resonances are apparent on the frequency response curves. The resonances are, depending on the animal, more or less pronounced. As a result of the improved accuracy changes of the malleus vibration response with time could clearly be discriminated. These changes can be related to shifts in frequency of the position of these resonances. Comparison of experimental frequency response and lumped parameter model predictions from literature shows that these resonances are not present in the model responses.

Volume displacement of the gerbil eardrum pars flaccida as a function of middle ear pressure.

The pars flaccida (PF) is a small region of the eardrum, with elasticity and histology completely different from the rest of the membrane, which has often been attributed a pressure regulating function for the middle ear (ME). In this paper, the volume displacement of the PF as a function of ME pressure is discussed. The deformation of the PF was measured in vitro in five Mongolian gerbil ears, by means of an opto-electronic moiré interferometer. Volume displacement was determined at small intervals in three sequential pressure cycles, in the range of +/- 0.4 kPa, +/- 2 kPa, and again +/- 0.4 kPa. The displacement was found to be a highly non-linear function of pressure, with a strong increase up to 0.4 kPa ME over- or underpressure and remaining nearly unchanged for pressures beyond 0.4 kPa. In all animals, maximal volume displacement was less than 0.5 microl, or 0.2% of total ME air volume. Clear hysteresis was found between the deformations at the same pressure level in the increasing and decreasing parts of the pressure cycles. Membrane behavior in the first 0.4 kPa pressure cycle was significantly different from that in the second 0.4 kPa cycle, which followed the 2 kPa pressure cycle. The results indicate that the ME pressure change regulation function of the PF is limited to very small pressure changes of a few hundred Pa around ambient pressure, and that larger ME pressures cause at least short-term changes in the membrane's behavior.

Malleus vibration mode changes with frequency.

The mode of vibration of the cat manubrium is investigated by measuring its vibration in response to sound stimulus at four locations between the umbo and the processus lateralis with a heterodyne interferometer. The determination of mode requires high precision in measurement because amplitude differences between the points are small (about 20% at low audio-frequencies). Changes in the frequency response with time have been reported in an earlier paper. The nature and magnitude of this time change is analysed in detail: over a period of 1 h the average change in amplitude is about 5% and in phase 5 degrees. The malleus vibration at some frequencies is purely translational, it is rotational at others and mixed at most frequencies. When the motion is rotational the position of the axis of rotation shifts with frequency, the shifts are so large that the axis can lie near the umbo so that amplitudes at the processus lateralis are larger than at the umbo. The classical concept of the malleus rotating around a fixed axis running from the anterior mallar to the posterior incudal ligament fits our measurements only at low frequencies.

Shape and derived geometrical parameters of the adult, human tympanic membrane measured with a phase-shift moiré interferometer.

The shape of the tympanic membrane is fairly complex and seems to be of significant importance in the coupling of the acoustic sound pressure in the external ear canal to the motion of the middle ear ossicles. A moiré shift interferometer was used to measure with great precision the shape of the external surface of human tympanic membrane. The dense matrix of z(x,y) values thus obtained is used to calculate different geometrical parameters (area, curvature, ...). We show further how the same data can be used to specify exactly the shape of the tympanic membrane in a mathematical finite-element model of the middle ear.

Measurement and modeling of boundary shape and surface deformation of the Mongolian gerbil pars flaccida.

The shape of the pars flaccida (PF) boundary and its pressure induced deformation was measured in five Mongolian gerbil ears, using an opto-electronic moiré interferometer. To determine the PF boundary, membranes were deformed by middle ear (ME) pressures of -2 kPa and +2 kPa. The boundary of the PF was defined as the locus of points where the pressure induced deformation begins. To a very high precision, this boundary was found to be a circle with the same radius for both over- and underpressure deformations. Between animals the radius varied from 0.760 mm to 0.778 mm. We show that the shape of the PF, while being deformed by static pressure in the ME, can be modeled as part of a sphere. Volume displacement can then be calculated as the volume enclosed by the sphere cap, delimited by the circular PF boundary plane. Volume displacement was calculated for membranes deformed by 400 Pa ME overpressure and -400 Pa ME underpressure. The agreement of the experimental data with the sphere cap model is shown to be very good, and results in a volume displacement measuring accuracy which is better than 10%.