INTRODUCTION

The streaked tenrec (Hemicentetes semispinosus) has an extraordinary mechanism of sound communication that consists of hard, keratinous quills in the mid-dorsal region (Gould, 1965; Eisenberg and Gould, 1970; Novak, 1999). It has been suggested that vibration of these quills acts as a sounding apparatus in this species, allowing communication between the mother and young (Eisenberg and Gould, 1970; Stephenson, 2003). Certainly vibration of quills produces sound in living individuals; however, the functional-morphological mechanism of these vibrations remained unclear. The rarity of the streaked tenrec has prevented detailed study of its skin and subcutaneous structure, and no descriptive or functional-morphological data have been published regarding the sounding organ. In this study, we made macroscopic and microscopic morphological observations on this sound communication system. We also used immunohistochemical methods to identify the type of muscular myosin associated with the vibrating motor of the quills, since only fast myosin could produce the necessary quick, strong vibrations of the quill vibrator disc (QVD).

MATERIAL AND METHODS

Microscopic and Immunohistochemical examinations

The area of thick cutaneous musculature on the reverse side of the skin was named the “quill vibrator disc” (QVD). Since the QVD area corresponds to the region of the quills, morphological relationships between the quills and QVD were examined by light microscopy. The muscle tissues of the QVD were excised from the reverse side of the skin, post-fixed in Bouin's fixative for 2 hours, and washed in 70% ethanol. The tissues were dehydrated in ethanol and embedded in paraffin at 65°C. Tissue blocks were sectioned transversely and horizontally at 4 µm thickness. After removing the paraffin with xylene, sections were stained with haematoxylin and eosin for microscopic observations, For immunohis-tochemistry, sections were incubated in 3% H₂O₂ dissolved in methanol for 30 min to quench endogenous peroxidase activity and stained by using the avidin-biotin-peroxidase complex method (Hsu et al., 1981) with the monoclonal antibodies against fast (Sigma, Saint Louis, Missouri, USA, Product No, M4276, clone MY-32, 1:8000 dilution) and slow (Sigma, Saint Louis, Missouri, USA, Product No, M8421, clone NOQ7.5.4D, 1:400 dilution) skeletal myosin, After an overnight incubation at 4°C with the primary antibodies, sections were incubated with biotinylated anti-mouse immunoglobulin (Nichirei, Tokyo, Japan) for 30 min. Following this step, the avidin-biotin-peroxidase complex was applied to the sections for 5 min, Immunoreaction products were obtained by subsequently incubating the sections in a diaminobenzidine-H2O2 solution. Staining controls included omission of the primary anti-serum. Tissues from the cranial, middle, and caudal parts of the QVD were compared immunohistochemically to identify similarities or differences in the position of the musculature.

Table 1.

Biological data for the four individuals examined in this study. QVD, quill vibrator disc; ND, no data were recorded.

Fig. 1.

Dorsal aspect of an adult streaked tenrec weighing 115.2 g. The quills are evident in the caudal part of the mid-dorsal area (arrow). The head is at the top.

RESULTS

In the streaked tenrec, specialized sounding quills are generally concentrated in a small area in the mid-dorsal region of the caudal part of the trunk (Fig. 1). This species possesses hard spines and normal long hairs that are morphologically different from the sounding quills (Fig. 2). Specimens used in this study had a total of 15 or 16 quills (Fig. 2). Macroscopic observations detected three rows of quills in the mid-dorsal region. Five quills were arranged laterally along each side and flanked five or six or medial quills.

Fig. 2.

Dorsal aspect of the quills, in the same individual as in Fig. 1. Sixteen light-brownish quills with circumferential dark step lines are concentrated in the caudal part of the mid-dorsal area. Five quills are arranged in the left lateral area (small arrows), and the group in the midline is evident (large arrows). Hair and spines have been partially cut away around the region to clearly show the quills. The cranial direction is to the left.

Fig. 3.

Dorsal aspect of a juvenile streaked tenrec weighing 21.0 g. Five quills are arranged on the left side (small arrows), and the other groups in the midline and on the right side are evident (large arrows). Hair and spines have been partially cut away around the region to clearly show the quills. The cranial direction is to the right.

Quills consisted of a hard, keratin-like spiny structure. They were entirely light brown, except for the surface in the basal half of the quills, which had ten darker, circumferential step lines (Fig. 2). The quill points were sharp, fine, and devoid of darker lines. The basal parts of quills in the lateral area were thicker than those in the medial region in the adult streaked tenrec we examined.

Table 1 gives the number, length, and diameter of quills, along with data on normal hard spines. Quills in juveniles were similar in length to those in adults, but were much smaller in diameter. The arrangement of quills in juveniles was similar to that in adults, comprising three rows in the midline and the adjacent areas bilaterally (Fig. 3).

The quill vibrator disc (QVD) (Fig. 4) had a craniocaudally elongated oval shape at the macroscopic level (Fig. 5). The area of the structure corresponded exactly to the area in which the sounding quills occurred. The muscular direction was longitudinal or corresponded to the margin of the QVD (Fig. 5). The size of QVD is indicated in Table 1. In the adult individual, the QVD was 16.8 mm in craniocaudal length and 8.55 mm in transverse width. The size of the QVD increased with increasing body size (Table 1).

Fig. 4.

Reverse side of the skin in the adult specimen. The QVD (arrows) appears elliptical and shows thick cutaneous muscles. The cranial direction is toward the top.

Fig. 5.

Elongated oval shape of the thick longitudinal musculature of the cutaneous muscles evident at the macroscopic level in the QVD of the adult specimen. The cranial direction is to the right.

We observed transverse (Figs. 6, 7) and horizontal (Figs. 8, 9) sections of the musculature. Muscle bundles consisted of thin, longitudinally curved skeletal muscle. Some muscle bundles and the accompanying connective tissues composed the elliptical tissue structure of the QVD. The elongated oval structure was symmetrical in the sagittal plane, and thin connective tissues divided the muscular structure into two lateral parts (Fig. 6). Striated myocytes containing oval-shaped nuclei formed the musculature of the QVD (Figs 7, 9). The basement membrane was located in the ventral area of the QVD (Fig. 6).

Fig. 6.

Transverse section of the QVD, stained with hematoxylin and eosin, showing the left (L) and right (R) sides of the ellipse of the QVD separated by thin connective tissue (large arrow). The structure is symmetrical in the sagittal plane on each side of this connective tissue, The basement membrane (small arrows) is evident in the ventral area of the OVD. Dorsal is toward the top and the left lateral direction is to the left, Scale bar: 100 µm.

Fig. 7.

Transverse section of the QVD, stained with hematoxylin and eosin, The musculature consists of many myocytes, each containing an oval nucleus peripherally, Scale bar: 30 µm.

Fig. 8.

Frontal section of the QVD, stained with hematoxylin and eosin. The muscles are divided into muscle bundles by thin connective tissue, Since the musculature is slightly dorsoventrally curved, an oblique section of each muscle is evident in the exactly frontal section. Apocrine sweat glands (arrows) are evident among the muscles, Scale bar: 100 µm.

Fig. 9.

Frontal section of the QVD, stained with hematoxylin and eosin. Myocytes comprise the longitudinal musculature. An oval nucleus is evident peripherally in each myocyte, Scale bar: 30 µm.

In the dorsal layer of the QVD, we observed sweat glands and hair follicles among the muscle bundles (Fig. 10). Apocrine sweat glands were concentrated in particular areas (Fig. 11). Arrector pilorum muscles were attached to hair follicles (Fig. 10).

Immunohistochemical reactions against fast myosin were observed largely in striated myocytes in the QVD musculature (Figs. 12, 13), whereas no reaction was observed against slow myosin (Fig. 14). Reactions within myocytes were diffuse (Fig. 13), and no structures except the musculature reacted (Fig. 12). There was no difference in immunoreactivity among individuals or in sampling positions in the QVD.

Fig. 10.

Apocrine sweat glands (large arrow) and a hair follicle (H) are evident among the muscle bundles. Arrector pilorum muscles (small arrows) are attached to the hair follicle. Stained with hematoxylin and eosin. Scale bar: 100 µm.

Fig. 11.

Apocrine sweat glands among the musculature. Stained with hematoxylin and eosin. Scale bar: 30 µm.

Fig. 12.

Transverse section of the QVD, at a sectional angle similar to that in Fig. 6. Fast myosin is distributed largely in the myocytes, indicated by immunostaining, which is not evident in any structures except the musculature. Dorsal is toward the top, and the left lateral direction is to the left. Scale bar: 100 µm.

Fig. 13.

Diffuse immunostaining within myocytes of the QVD. No reaction is evident in the nuclei. Scale bar: 30 µm.

Fig. 14.

No immunostaining for slow myosin is evident in the musculature, Scale bar: 30 µm.

DISCUSSION

Renrecs are noteworthy for their phylogenetic position showing the parallel evolution of the Lipotyphla (Asher, 1999; Asher et al., 2003), and the functional-morphological convergence (for example, in limbs as the locomotor apparatus) between these two phylogenetic groups has been examined in detail and discussed (Endo et al., 2006, 2007). The quill vibration system of the streaked tenrec represents one of the most extraordinary sounding mechanisms in mammals. Sound communication and echolocation in mammals are generally realized by vocalization related to the air pathway. Species of Sorex, Neomys, and Diplomesodon in the Lipotyphla communicate intraspecifically by emitting vocal signals within their tunnels (Movchan and Shibkov AA, 1982). In species of Lipotyphla, sound communication may contribute to defense, attracting mates, and actively searching for moving prey. Why vocalization has not developed in tenrecs has remains unclear, but the quill vibration mechanism of the streaked tenrec may correspond functionally to vocal communication in the Lipotyphla. We suggest that sounding by quill vibrations may be reasonable for spiny animals such as the streaked tenrec, since hard, keratinous quills and the vibrating motor could have easily evolved from spines and cutaneous muscles, respectively.

From the present morphological findings, we conclude that the QVD is a specialized apparatus in the cutaneous muscle of the trunk. Arrector pilorum muscles are not functional-morphologically related to the control of the QVD, or to the movement of non-specialized hairs (Fig. 10). Since enlarged arrangements and fast movement of the musculature are needed to vibrate quills, we suggest that general arrector pilorum muscles cannot be adapted for this functional role. Although vibrations have not been analyzed by filming living animals, we suggest that the bases of the quills, with circumference step lines, may rub against each other to produce sound. The longitudinally and marginally running cutaneous musculature may effectively act as a rubbing motor for the three groups of quills arranged in the midline and bilaterally in the dorsal region. In the future, however, we need to explain functional-morphologically how sound is produced by the circumference step lines of the quills.

In this study, we examined the growth of quills by comparing adults with juveniles (Table 1). The number and arrangement of quills did not alter during growth. The length of quills did not increase during growth like that of the normal hard spines, whereas the quill diameter increased from 0.4–0.5 mm in juveniles to 0.8–0.9 mm in adults. Although differences in the vibrating function between juveniles and adults remain unclear, the restricted variation in quill number and early development of quill length suggest that sounds produced by quill vibrations may contribute to sound communication and echolocation even in juveniles. This is consistent with data demonstrating that sounding is important in helping the streaked tenrec mother and young locate one another (Eisenberg and Gould, 1970; Novak, 1999).

The QVD is also equipped with apocrine sweat glands. This suggests that the skin surface near the QVD may secrete exocrine chemical signals related to the sense of smell. Our results confirm the presence of systems contributing to non-visual communication via the QVD in this species.

The structure of the cutaneous musculature cannot be readily analyzed. Our study was related to work done on the sugar glider (Petaurus brevicepis) (Endo et al., 1998), which indicated that observations made skillfully from the reverse side of the skin provide macroscopic data on the cutaneous muscles. We used similar techniques in our observations of the QVD in the streaked tenrec.