Abstract

A three-dimensional (3-D) model of the digestive system of Periplaneta americana was built for the first time based on hematoxylin and eosin (H&E) staining, the study of multiple cross-sections of the larval cockroach, and 3-D reconstruction technology. The digestive system of P. americana includes the foregut, midgut, and hindgut and takes up most of the celom. The foregut comprises almost one half of the digestive system (43.57%). The midgut, the critical region for digestion and absorption, has the second highest volume ratio (35.21%). The hindgut, with the lowest volume ratio (21.22%), includes the ileum, colon, and rectum. After the ileal valve is the colon. The 3-D model presented in this paper provides a stereoscopic view for studying the adjacent relationship and arrangement of different gut sections of P. americana.

The American cockroach, Periplaneta americana, is the dominant cockroach species in Latin America, Japan, and Taiwan (Arruda et al. 1995). In many studies, P. americana has been proven to contribute to asthmatic reactions in humans (Chapman et al. 1996, Arruda et al. 2001). Over the past decade, several cockroach allergens of P. Americana and other roaches have been identified, sequenced, and produced as biologically active recombinant proteins (Chapman et al. 1996, Wu et al. 1996, 2000). It has also been suggested that Per a 3, a major species-specific allergen of P. americana (Wu and Lan 1988, Wu et al. 2003, Gao et al. 2005), is possibly synthesized in and secreted from the epithelia of the midgut mucosa and excreted from the body in fecal pellets (Wu et al. 2007). Hence a better understanding of the P. americana digestive system may provide important information about the generation of this major allergen.

In this study, the three-dimensional (3-D) reconstruction technology was adopted to generate the 3-D model of the digestive system of the larval P. americana on the basis of serial paraffin-embedded cross-sections observed using light microscopy. Taking advantage of this technology, the accurate spatial position and morphology of different intestinal sections can be measured and described in a stereoscopic view. Thus, this study provides a new approach for exploring the internal structure of P. americana and gives insight into mechanisms of P. americana digestion.

Materials and Methods

Preparation and Observation of Serial Paraffin Slices.

Periplaneta americana animals were provided by the Allergy and Immunology Institute of Shenzhen University and maintained at room temperature in glass incubators. Larval P. americana were removed from the incubators, fixed in Bouin’s solution for 24 h, placed into a 1.5% agar solution, and left to spontaneously coagulate. Subsequently, the insect’s body was positioned in the center of the agar and dehydrated in graded ethanol and then xylene before being embedded in paraplast (Sigma, St. Louis, MO). Serial 10-μm sections were cut in a sledge microtome, and each slice was transferred to a glass slide coated with poly-l-lysine (Sigma) for drying on a hot plate at 37°C. After hematoxylin and eosin (H&E) staining, sections were observed with a light microscope (BX51; Olympus, Tokyo, Japan), and images were transferred to a PC using a digital camera (DP70; Olympus). The identification and terminology followed the description of Bell and Adiyodi (1981).

Three-dimensional Reconstruction and Measurement of the Digestive System.

Cross-sections were selected, prepared, and photographed at ×10 magnification. Images were treated using image analysis software Image-Pro Plus (IPP; Media Cybernetics, Bethesda, MD), which included brightness contrast control, edge sharpening, and image enhancement. Fourier transform was used to align 2-D images, including the translation and rotation of the rigid body. Areas of the digestive system were excised and overlaid to create an image stack. By defining the x, y, and z dimensions of the voxels that created an image volume, the 3-D model was synthesized, and the spatial measurements (i.e., distance, angle, and cross-sectional area) were determined. Both 2-D and 3-D morphology parameters of the sections and the 3-D model were measured using the same software.

Results

Our observations showed that the digestive system occupies a large part of the body cavity of P. americana. The alimentary canal was shown to be tubiform, with the length from mouth to anus twice as long as the length of the entire body. The foregut, midgut, and hindgut of the digestive system were visualized (Figs. 1 and 2B). We found that the foregut (mouth, pharynx, esophagus, crop, and proventriculus) takes up one half of the length of the digestive system, whereas the remainder is comprised of the midgut (midgut and digestive ceca) and hindgut (ileum, colon, and rectum).

Fig. 1

(A) Cross-section of proventriculus. Scale bar = 40 μm. (B) Cross-section of crop, proventriculus, and midgut. Scale bar = 40 μm. Sv, stomodeal valve. (C) Cross-section of ileum and colon. Scale bar = 40 μm. (D) Cross-section of rectum and rectal pad. Scale bar = 40 μm. Rp, Rectal pad. (Online figure in color.)

Fig. 1

(A) Cross-section of proventriculus. Scale bar = 40 μm. (B) Cross-section of crop, proventriculus, and midgut. Scale bar = 40 μm. Sv, stomodeal valve. (C) Cross-section of ileum and colon. Scale bar = 40 μm. (D) Cross-section of rectum and rectal pad. Scale bar = 40 μm. Rp, Rectal pad. (Online figure in color.)

Fig. 2

(A) Computer 3-D reconstruction map of a larval P. americana (transparent mode of outside contours; left view). Mo, mouth; Ph, pharynx; Es, esophagus; Cr, crop; Pr, proventriculus; Mg, midgut; Il, ileum; Co, colon; Re, rectum. (B) Computer 3-D reconstruction map of the rotation of a larval P. americana (transparent mode of outside contours; ventral view) in 3-D space. (C) Incision of the computer 3-D reconstruction model (X, Y, and Z axis). (Online figure in color.)

Fig. 2

(A) Computer 3-D reconstruction map of a larval P. americana (transparent mode of outside contours; left view). Mo, mouth; Ph, pharynx; Es, esophagus; Cr, crop; Pr, proventriculus; Mg, midgut; Il, ileum; Co, colon; Re, rectum. (B) Computer 3-D reconstruction map of the rotation of a larval P. americana (transparent mode of outside contours; ventral view) in 3-D space. (C) Incision of the computer 3-D reconstruction model (X, Y, and Z axis). (Online figure in color.)

IPP and human-computer interaction allowed arbitrary rotation of the reconstructions and random virtual sectioning (Figs. 2B and C), which made it possible to observe the configuration, location, and adjacent relationship of individual gut regions in the intact cockroach. The area, perimeter, and length measurements obtained from serial sections of the gut region and the associated statistical findings are summarized in Table 1. The volume and length of each gut region from the reconstructed model after sectioning are tabulated and summarized in Table 2.

Table 1

Measurement of 2-D parameters of P. americana (mean ± SD)

Table 2

Measurement of 3-D parameters of P. americana

Discussion

Studies have focused on the digestive system or allergens of P. americana. Purification, properties, and substrate specificity related to a digestive trypsin from P. americana adults have been clarified (Lopes and Terra 2003). Also, cloning, expression, and structure prediction of the CR-pIIgene from P. americana and the identification of the major allergen in P. americana by high-performance liquid chromatography-electrospray-ionization tandem mass spectrometry (HPLC-ESI-MS-MS) have been done (Hu et al. 2005, He et al. 2006). Few studies explored both the digestive system of P. americana and its association with allergies because it is not convenient to incise the gut to label allergens. The 3-D model of the digestive system of P. americana could solve this problem. The results of this study show detailed and precise measurements of the different parts of the P. americana’s digestive system. Therefore, using immune electron microscopy may further explore the ultrastructural location of allergens in P. americana and study the relationship between allergen secretion, release, and allergen-related diseases, which would provide guidance and reference for development of allergen vaccines (Bao et al. 2007, Liu et al. 2007).

Acknowledgements

We thank Q. Wang, B. Shi, and F. Lou for excellent technical assistance. Funding was provided by National Science Fund (30571625, 39860071) and Key Projects of Guangdong Province (2003 A3080502).

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