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First published online May 24, 2004
Journal of Experimental Biology 207, 2255-2265 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01004

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Dietary influences over proliferating cell nuclear antigen expression in the locust midgut

E. Zudaire¹, S. J. Simpson², I. Illa¹ and L. M. Montuenga^1,*

¹ Department of Histology and Pathology, Schools of Medicine and Sciences, University of Navarra, E-31080 Pamplona, Spain
² Department of Zoology and University Museum of Natural History, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

View larger version (122K): [in a new window]   Fig. 1. PCNA-like immunoreactivity throughout the locust midgut. (A) Panoramic view of locust midgut caeca walls. Strong immunoreactivity appears mainly in the core of the regenerative nests (arrows). (B) Ventricle wall. PCNA-like immunoreactivity is exclusively present in the cells of the regenerative nests at the base of the epithelium (arrows). Enterocytes surrounding the regenerative nests are progressively less stained, and no immunoreactivity is found in mature enterocytes outside the nests (arrowheads). (C) PCNA-like immunoreactivity in the locust ampullae of Malpighian tubules. PCNA-like immunoreactivity is found in clusters of regenerative cells (arrows). (D) PCNA-like immunoreactivity and BrdU-immunoreactivity in serial consecutive sections of the midgut in the caeca region. (E) Western blot analysis of locust gut tissue extract using antiserum against PCNA. A single band of 30 kDa is detected under reducing conditions. Lane 1, molecular mass markers; lane 2, locust gut extract; lane 3, negative control without antibody. Scale bars, 25 µm in A and 50 µm in B–D.

View larger version (18K): [in a new window]   Fig. 2. Effect of microwave heating time on PCNA-like immunoreactivity in the nests of midgut caeca. Immunoreactivity is only noticeable after 20 min of microwave heating, and the optimum staining is reached after 40 min of microwave treatment. However, longer exposures to microwave heating dramatically drop the staining levels below the optimum.

View larger version (19K): [in a new window]   Fig. 3. Differences in PCNA-like immunoreactivity (bars, PCNA-I; line, PCNA-OD) between the five regions of the locust midgut. For statistical analysis, values of caeca have been compared to the rest of the regions. There are no significant differences between the values of caeca and ampullae. Caeca and ampullae of the Malpighian tubules show higher levels of staining compared with ventricle (*P<0.05, **P<0.01, ***P<0.001).

View larger version (49K): [in a new window]   Fig. 4. Effect of diet composition, sex and time during the fifth instar on values for PCNA-I (A) and PCNA-OD (B). The balanced diet (21:21) shows the highest levels of PCNA, and as the diets became more unbalanced in their ratio of protein to digestible carbohydrate, the level of PCNA dropped. Males showed more PCNA than females, and PCNA levels were higher on day 4 than on day 1.

View larger version (16K): [in a new window]   Fig. 5. Regression lines (PCNA-I, solid line; PCNA-OD, broken line) showing the relationships between both PCNA-I (squares) and PCNA-OD (circles) values in locusts fed on a given diet and the relative distance of this diet from a nutritionally optimal composition (see Chambers et al., 1995) on days 1 (A) and 4 (B). Regression lines: (A) PCNA-I, y=–0.5x+18.64, r2=0.23; PCNA-OD, y=–3.33x+ 172.1, r2=0.62; (B) PCNA-I, y=–1.26x+31.61, r2=0.25; PCNA-OD, y=–2.94x+180.8, r2=0.6.

View larger version (26K): [in a new window]   Fig. 6. Plot showing the values of PCNA-I (squares) and PCNA-OD (circles) related to the quantity of food eaten (diet 7:7, open bars; diet 21:21, solid bars) through the first 8 days of the fifth instar for both diet 7:7 (lines) and diet 21:21 (dotted lines). Both PCNA-I and PCNA-OD values increased between day 0 and day 3 and decreased thereafter. Notice the correlation between the amount of food eaten and the levels of both PCNA-I and PCNA-OD.

View larger version (18K): [in a new window]   Fig. 7. Plot showing the regression lines between values for PCNA-I and PCNA-OD (A for diet 7:7 and B for diet 21:21) and the quantity of 7:7 and 21:21 diet eaten by locusts across the first 8 days of the fifth instar. Regression lines: (A) PCNA-I, y=21.47x+37.5, r2=0.81; PCNA-OD, y=40.12x+173.8, r2=0.55; (B) PCNA-I, y=34.1x+39.77, r2=0.65; PCNA-OD, y=98.28x+179.3, r2=0.62.

View larger version (24K): [in a new window]   Fig. 8. A frequency spectrum showing the integrated optical density (IOD) values (in arbitrary units) for Feulgen stains of the mature and undifferentiated nuclei of the caecal epithelial cells of locusts fed on different synthetic foods (N=400 in each group).