Proceedmgs of the 7th Iniernatumal Workmg Conference on Stored-product Protectum - Yolume 2

Radiation-induced changes in the midgut of insects, pests ofstored products

Maryla Szczepanik' and Stamslaw Ignatowicz'

Abstract

Degenerative changes m the midgut of msect larvae andadults of some stored product pests (Trogoderma grananumEv., Tnbohum confusum DuVal , Plodia mterpunctellaHubner) are positively correlated with both the dose andtime elapsed after irradiation exposure. Therefore, apathological syndrome of irradiation effects on the midgutmay be used as an efficient method for Identification ofirradiated msects When the destruction of regenerativenidi, lack of brush border, and vacuolated epithehal cells areobserved within the transection of the midgut, then one maysuspect that the pest was irradiated a few days ago Whenthe total disintegration of the midgut IS observed, one mayconclude that the pest was irradiated WItha lugh dose a fewdays ago, or WIth a low dose, but several days agoThIS test ISquick and convement when a rapid processmg

techmque mvolvmg 1, -l-dioxane and Polybed 812 fordehydrating and embeddmg the midgut IS used Usmg tlustechnique, a tramed person is able to process the msectrrudgut from fresh tissue to sectionable embedded blockswithin 5 hours The test based on the pathological changesmduced by Irradiation m the msect midgut may Identifyconsistently either irradiated larvae or adults

Introduction

Insects have only a few cells that have to undergo division, aprocess known to be highly susceptible to damage byexposure to irradiation Some of these cells are placed m theepithehum of the midgut, where they renew cellsresponsible for digestion and absorption of the products ofdigestion. DIVISIonof epithelial cells occurs as new cellsgrow m to replace old worn-out cells m a very metabohcallyactive tissueThere IS mterest wlthm the food mdustry and SCIentific

commumty m easy to apply techmques to venfy whether

1 N CopernlCUSUmverslty, Department of Invertebrate Zoology,Torun; Poland

2 Warsaw Agncultural Umverslty, Department of AppliedEntomology, Warsaw, Poland

hvmg msects found in a shipment have or have not beenirradiated (IAEA 1992)Therefore, the objective of our work has been to study

whether radiation-mduced changes in the midgut of msectsmay be used to detect some stored product pests irradiatedfor quarantme security.

Material and Methods

Adults of the confused flour beetle, Tnbolium confusumDuVal, and larvae of the khapra beetle, Trogodermagrananum Ev, and, the Indian meal moth, Plodiamterpunctella Hubner, were collected from laboratorycultures Insects were Irradiated m a Co-60 irradiator WItho .0 (control), O.1, 0 .3 and 0 5 kGy dose of gammaradiation (dose rate = ca. 20 Gy/mm) After thetreatment, the treated and untreated (control) msects werekept in separate jars WIthwheat gram for 1, 2 or 3 weeks.After this period, msects were prepared for histologicalanalysis Two histological methods were used: (a) astandard method, and (b) a fast method.The standard method was used only m studies with adults

of the confused flour beetle (FIgS 3, 6, 9 and 11) Beetleswere fixed WIth 2.5 % buffered glutaraldehyde for 3 hrs,usmg 0.1 M cacodylate buffer, pH 7.2 Insects were thenwashed several times WIth the same buffer, dehydrated m astandard alcohol senes and acetone, and embedded mSpurr's resm.For the second method, the buffer and fixative were

prepared accordmg to the formulas descnbed by Hayat(1970) Plastic embeddmg mixture was prepared fromcomponents purchased from Polysciences Inc. (Warrington,PA) accordmg to the schedule for embeddmg suggested byShearer and Hunsicker (1980) A small part of msect larvaem which the midgut ISlocated was cut and fixed for 30 mmm Kamovsky's fixative at room temperature, and thennnsed three tImes, for 2 mm. each step, m 0.1 Mcacodylate buffer (pH = 7.4) Next, tissues were qmcklydehydrated m 70% ethanol and m 1,4-dIOxane, mfIltrated mthe resm/dIOxan mIxture and pure resm. Mter mfiltratIonthe matenal was transferred to capsules contalmngembeddmg mIxture Polybed 812, and It was polymenzed at80°C for 3 hoursTransverse semI-thm SEctionswere cut on the LKB-8800

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Proceedings of the 7 th Intemational Wm'lcingConference on Stored-produet Protection - Volume 2

microtome, mounted on slides, stained with toluidine blue,and observed under a light microscope.

Results

Normal structure of the midgut

Insect midgut is the main site for digestion and absorptionof the products of digestion, and is a very metabolicallyactive tissue. The midgut consists of single layer ofepithelium placed on a basement membrane. The epitheliumis made up of columnar cells, i. e. , secretory cells. Theseepithelial cells are relatively high and form a regular andcompact wall. An oval nucleus is located in the central partof each columnar cell. The apical surface of each columnarcell bordering with the gut lumen is covered with microvillaewhich create the tight structure called the brush border.The regenerative cells are another type of cells within the

epithelium. These tiny cells form the regenerative nidi thatare regularly located at the base of the columnar cells (Figs1 and 2). In the midgut of T. confusum adults, theregenerative cells are located in the evaginations of themidgut wall, forming paunch-like diverticula known as theregenerative crypts (Fig. 3).In the epithelium of P. interpunctella larvae, the goblet

cells are interspersed among the columnar cells. Thecytoplasm of these pear-shaped cells is reduced, and the

apical border of the cell surface invaginates to form a deepcavity. In this cavity there are numerous cytoplasmicextensions. Flat nucleus of the goblet cell is located basally,below its cavity.The epithelium rests on well-developed basement

membrane that is surrounded by a layer of circular musclesand an outer longitudinal muscle coat.

Changes in the midgut induced by irradiation

The following gross changes in the midgut of insects werefound. On the 7th day after irradiation with a dose of 0.3kGy, the epithelial cells of larvae of the Indian meal mothwere elongated and vacuolated. Their nuclei were distinctlyenlarged. Brush border was seen on a large surface ofepithelial cells, but it disappeared on the most affected cells.Cytoplasmic extensions of the goblet cells weredegenerated, and their fragments were often noted in thecavity of goblet cells. All regenerative cells were lost, andthe basement membrane formed many folds as a result of themuscle contraction (Fig. 4). At the same time, the midgutof the T. granarium larvae treated with a dose of 0.3 kGywas much more affected. The regular structure of theepithelium was disturbed. Most of columnar cells elongatedinto the gut lumen, and their apical part swelled distinctly.Cell nuclei were enlarged, and they moved into the directionof the gut lumen. The basement membrane formed largefolds as a result of the distinct muscle contraction (Fig. 5).

Fig. 1. Transection of the midgut of control larva of the Indian meal moth, P. interpunctella (x370). L- gut lumen, BBbrush border, GC- goblet cells, CC- columnarcellsof the epithelium, ML - muscle layer, BM- basementmembrane,RN - regenerative nidi.

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Proceedings of the 7 th International Working Conference on Stored-prodj«:t Protection - Vol:wme2

Fig. 2. Transection of the midgut of control larva of the khapra beetle, T. granarium ( x 320). L - gut lumen, BB - brushborder, CC- columnar cells of the epithelium, RN - regenerative nidi, BM - basement membrane, ML- muscle layer.

Fig. 3. Transection of the midgut of control of the confused flour beetle, T. confusmn ( x 4(0). L - gut lumen; BB - brushborder, CC- columnar cells, N - nuclei, RC- regenerative crypt, ML- muscle layer.

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Fig.4. Transection of the midgut of P. interpunctella larva irradiated with 0.3 kGy dose; the section has been done on the 1stweek after treatment ( X 370). L - gut lumen, EC - epithelial cells elongated into the gut lumen, CC - vacuolatedcolumnar cells, N - enlarged nuclei, BE - brush border, CE - fragments of degenerated cytoplasmic extensions in thecavity of the goblet cells, ML- muscle layer.

Fig. 5. Transection of the midgut of T. granarium larva irradiated with 0.3 kGy dose; the section has been done on the 1stweek after treatment ( X 400). L - gut lumen, CC - enlarged and vacuolated columnar cells of the of the epithelium, V- vacuoles, :rvtL - muscle layer.

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On the 14th day after irradiation treatment with a dose ofo . 3 kGy, the midgut of all insects studied was severelydisrupted. Disintegration process seemed to occur faster inbeetles than in moth larvae. Changes in the midgut ofbeetles induced by irradiation were similar. All columnarcells wer~ distinctly swelled and vacuolated, and filled upthe gut lumen together with the cellular debris (Fig. 6) orwere located on folded basement membrane (Fig. 7).Within these cellular debris, the regenerative cells were notvisible, but some spherical and swelled cell nuclei werefound. The muscle layer was distinctly thickened and formednumerous folds. Changes in the midgut of the Indian mealmoth larvae progressed slowly. On the 14th day afterirradiation treatment with a dose of 0.3 kGy, the epitheliumof their midgut was lesser affected than in beetles. Althoughthe irradiation caused a severe damage to the columnarcells, fragments of the epithelium were preserved, and thegoblet cells were found to be interspersed among undamagedcolumnar cells (Fig. 8).The highest dose used (0.5 kGy dose) caused the most

severe damage to the insect midgut. On the 14 th day afterirradiation treatment, the columnar cells completelyhistolysed, and as a result the epithelium disappeared andthe gut lumen enlarged considerably. The basementmembrane formed large folds as a result of the distinctmuscle contraction. Only a few cellular debris were found atthe muscle layer (Figs 9 and 10). The same changes werefound in the insect midgut on the 21st day after irradiationtreatment, when a dose of 0.1 kGy Vias used (Fig. 11).

Discussion

Results obtained confirm the previous findings (Ashraf et a1.1971, Brower and shraf 1972, Jafri and Ismail 1977,Quereshi et a1. 1975, Riemann and Flint 1967, Vinson eta1. 1969) concerning the midgut of irradiated larvae oradults belonging to the insect orders of Coleoptera,Lepidoptera and Orthoptera.Changes in the midgut structure observed in irradiated

larvae were: (1) elongation and swelling of the columnarcells of the epithelium; (2) vacuolisation of the epithelialcells: (3) loss of the brush border; (4) loss of epithelial cellboundaries: (5) damage to epithelial cell nuclei (chromatingrains in the midgut lumen); (6) disruption of theepithelium: and (7) thickening of muscles and theircontraction resulting in many foldings of the midgut.Undifferentiated cells of the midgut forming the

regenerative nidi of the T. granarium larva were found to bethe most sensitive to irradiation. Damage to them resultedin the total disruption of the epithelium by preventing thereplacement of the secretory cells exhausted by secretoryactivity. Degree of damage to the regenerative nidi seems tobe dependent on the dose of radiation, time elapsed after thetreatment, and on the pest susceptibility to irradiation.Lepidopterans are known to be more radiation-resistant thanbeetles, therefore radiation-induced changes in the midgutepithelium of the Indian meal moth larvae progressed slowerthan in the coleoptarans.

FIg. 6. 'Iransection of the midgut of T. oonfusumirradiated with 0.3 kGy dose; the section has 1=1done on the 2nd week after treatrrent(x400). L- gut lumen, EC- vacuclized epithelial cells, N - nuclei, 8M-basement membrane, ML - muscle layer.

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Proceedings of the 7 th International Working Conference on Stored-product Protection - Volurne 2

F1g. 7. Transection of the midgut of T. granarium larva irradiated with 0.3 kGy dose; the section has been done on the 2ndweek after treatment ( X 400). L - gut lumen, CE - vacuolated cells of epithelium, N - nuclei in the gut lumen, BM-basement membrane, ML - muscle layer.

Fig. 8. Transection of the midgut of P. interpunctella larva irradiated with 0.3 kGy dose; the section has been done on the 2ndweek after treatment ( X 370). L - gut lumen, VC - vacuolated cells of the epithelium, N - nuclei of the epithelial cellsscattered within the gut lumen, GC - intact goblet cells, ML- muscle layer.

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Fig. 9. Transection of the midgut of T. granarium larva irradiated with 0.5 kGy dose; the section has been done on the 2ndweek after treatment (x 400). L ~ gut lumen, EP ~ fragments of disrupted epithelium, N ~ nuclei of epithelial cells,ML muscle layer, FT~ fat body.

Fig. 10. Transection of the midgut of T. confusum irradiated with 0.5 kGy dose; the section has been done 2nd week aftertreatment ( X 400). L - gut lumen. EP - a part of disappearing epithelium, ML - muscle layer.

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Proceedings ~fthe 7th Internatirmal Working Conference on Stored-product Protection. - Voh,me 2

Fig. 11. Transection of the midgut of 1'. confusum irradiated with 0.1 KGydose; the section has been done 3rd week ( x400). 10-gut lumen, ML- muscle layer.

These histopathological changes in insect midgut arepositively correlated with both dose and time elapsed afterirradiation exposure. Therefore, the pathological syndromeof irradiation effects on the midgut may be used for anefficient method of identification of irradiated insects.An ideal method for detection of irradiated insects should

be: (1) specific for irradiation and not influenced by otherprocesses, (2) accurate and reproducible, (3) have adetection limit below the minimum dose likely to be appliedto agricultural commodity as a quarantine treatment, (4)applicable to a range of pests, (5) quick and easy toperform, and (6) capable of providing an estimate ofirradiation dose (IAEA 1992). The histopathological methodfor identification of irradiated insect pests fulfills the most ofthese requirements (Szczepanik and Ignatowicz 1994,1995a, b).When the destruction of regenerative nidi, lack of brush

border, and vacuolated epithelial cells are observed withinthe transection of the midgut, then one may suspect that thepest was irradiated a few days ago. When the totaldisintegration of the midgut is observed, one may concludethat the pest was irradiated with a high dose a few days ago,or with a low dose, but several days ago.Physical causes other than ionizing radiation producing

disruption of the epithelium in the insect midgut as a resultof destruction of regenerative nidi are unknown. Therefore,the histopathological method for identification of irradiatedinsect pests seems to be specific for irradiation and not

influenced by other processes. The method has a detectionlimit below a 0.3 kGy dose, the minimum dose likely to beapplied to agricultural commodity as a quarantine treatment.The method seems to be applicable to a wide range of pests,as the similar histopathological changes were found inirradiated larvae or adults of T. confusum, Dermestesmaculatus DeGeer, Tenebrio molitor L., Anthonomusgrandis Boh. , Schistocerca gregaria L., P. interpunctella,Heliothis virescens Schiff. , and others. The method may bequick and easy to perform, when a rapid processingtechnique for dehydrating and embedding the larval midgutinvolving 1, 4-dioxane and Polybed 812 is used. Using thistechnique, it is possible to process the insect midgut fromfresh tissue to sectional embedded blocks within 5 hours(Shearer and Hunsicker 1980, Szczepanik and Ignatowicz1995a, b). However, more studies are needed to indicatesome specific post-irradiation changes in the midgut, whichare capable of providing an estimate of irradiation dose used.

Acknowledgments

Research was carried out with the financial support of theInternational Atomic Energy Agency in Vienna (ResearchContract No. 6942/RB). The authors acknowledge thefinancial support.

References

Ashraf, M., Brower, r.n., Tilton, E. W. 1971. Effect of1109

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gamma radiation on the larval midgut of the Indian mealmoth, Plodia interpunctella (Lepidoptera: Phycitidae ):Radiation Research, 45, 349 - 354.Brower, J. H., Ashraf, M. 1972. Histopathology ofirradiated yellow meal worm adult and larval midguts.Annals of the Entomological Society of America, 65, 950-954.Hayat, M. A. 1970. Pnnciples and Techniques of ElectronMicroscopy: Btological Applications, vol. 1. Van NostrandReinhold Co. , New York.IAEA. 1992. Use of Irradiation as a Quarantme Treatmentof Food and Agncultural Commodities. InternationalAtomic Energy Agency Panel Proceedmgs Series, Vienna.Islam, S., Rahman, R. 1996 Gamma radiation mducedhistopathological changes in larval midgut of the hidebeetle, Dermestes maculatus DeGeer (Coleoptera,Dermestidae). Polskie Pismo Entomologiczne, 65.Jafri, R. H., Ismail, M. 1977. Cytological lesions in themidgut of Tnbolium confusum larvae exposed to gammaradiation Journal of Invertebrate Pathology, 29, Id - 17.Ouereslu, S.A , Mohiuddm, S. , Akram, N. 1975.' Effectsof gamma radiation and hempa on the midgut of the desert,locust Schistocerca gregaria. Foha Biologica, 23, 123 - '127.Riemann, J. G. , Flint, H. M. 1967 Irradiation effects onmidgut and testes of adult boll weevil, Anthonomus

grandis, determmed by histological and shieldmg studiesAnnals of the Entomological Society of America, 60, 298-308.Shearer, T. P , Hunsicker, 1.G. 1980. A rapid method forembeddmg tissues for electron microscopy usmg 1, 4-dioxane and Polybed 812. Journal of the Histochemistryand Cytochemistry, 28, 465 - 467.Szczepamk, M., Ignatowicz , S. 1994. Identification ofirradiated msects: Changes m the midgut of the confusedflour beetle, Tribolium confusum DuVal., induced bygamma radiation. Roczmki Nauk Rolniczych, Sena E, 24,69-77.Szczepamk, M. , Ignatowicz , S. 1995a. A rapid method ofdetection of irradiated insects: Changes in the midgut oflarvae of the Indian meal moth, Plodia interpunctellaHubner, mduced by irradiation. Bulletm of the PohshAcademy of Sciences, Biological SCiences, 43, 121-126.Szczepanik, M , Ignatowicz , S. 1995b. Changes in themidgut oflarvae of the khapra beetle, Trogodermagrananum Everts, mduced by gamma radiation. Bulletm ofthe Polish Academyof SCiences, BiologicalSciences, 43 (3-4): 205-210.

Vinson, S. B., Londono, R. 1., Bartlett, A. C 1969.Effect of gamma radiation on tissues of the tobaccobudworm, Hehothis virescens. Annals of the EntomologicalSociety of America, 62, 1340 - 1347.

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