Volatile fatty acids and methane production in relation to anaerobic carbohydrate fermentation in Oryctes nasicornis larvae (Coleoptera: Scarabaeidae)

0022-1910/80/1201-4819

J. Insecr Phvsiol., Vol. 26, pp. 819 to 828. Eli”, Pergamon Press Ltd. 1980. Printed in Great Britain.

SOZ.OO/O

VOLATILE FATTY ACIDS AND METHANE PRODUCTION IN RELATION TO ANAEROBIC CARBOHYDRATE FERMENTATION IN OR YCTES NASICORNIS LARVAE (COLEOPTERA: SCARABAEIDAE) COLETTE BAYON Equipe de Recherche Associee au C.N.R.S N”231, Laboratoire de Zoologie, Facultt: Boulevard Gabriel, Universitk de Dijon, 21100 Dijon, France

des Sciences,

(Received 28 March 1980; revised 22 Ju1.v 1980)

investigations into the conditions of cellulose digestion by Oryctes nasicornis larvae show that the intestinal contents constitute a reducing alkaline medium. The mesenteron presents facultative conditions and high pH values whereas in the proctodeum anaerobiosis is strict and the pH approaches neutrality. No cellulase activity is detected either in the gut walls or the contents. Among the products of polysaccharide fermentation, volatile fatty acids (VFA) and methane are identified. The VFAs are 3 or 4 times more concentrated in the mesenteron than in the proctodeal dilation and acetic acid predominates. The VFAs, which are also present in the haemolymph, must cross the intestinal barrier. Methane formation takes place exclusively in the proctodeal dilation from which it is released to the exterior. Abstract-Physico-chemical

INTRODUCTION CERTAIN Scarabaeidae larvae have not only a xylophagous diet but also show a particular intestinal differentiation in having a very large proctodeal dilation (BUCHNER, 1953). WERNER (1926) demonstrated the degradation of ingested wood in the intestine of Potosiu and proposed the involvement of proctodeal bacteria in this process. Various bacteriologicalinvestigations [e.g. WIEDEMANN(1930) on Osmoderma, Cetonia and Oryctes, COUTURIER (1961) on Melolontha and MARSHALL(1963) on Oryctes] suggest that the proctodeal flora is cellulolytic. RIPPER (1930) on Dorcus and Cetonia, SCHLOTTKE (1945) on Oryctes and MARCUZZI and TURCHETTO-LAFISCA (1977) on Orvctes monoceros failed to demonstrate the existence of cellulase activity. So the problem as to whether the intestinal contents of Scarabaeidae larvae undergo cellulolytic processes remains unsolved. The present report, carried out on the larvae of Oryctes nasicornis, investigates the problem by characterizing the physico-chemical parameters of the gut contents, the enzymatic elements present, and by biochemical analysis of various substances which appear in the ingested food.

MATERIALS

to the natural diet and damp pure a-cellulose which allows the third instar larva to survive but prevents moulting or metamorphosis. Study of the physico-chemical

characteristics

Study ofthe H+ ion concentration of thegut contents.

Three methods were used. Dyes in alcohol solutions (25% ethanol), at 2% for bromothymol blue, thymol blue and neutral red and at 1% for bromocresol purple and phenol red. The observations were made in a small porcelain cup after mixing a little of the sample with 3 drops of indicator. Dyes in a 2% mixture with the wood-dung diet or with the pure a-cellulose. The larvae, reared on the stained foodstuffs, have a gut content originating entirely from these media after 24 hr. Under these conditions, the colour of the indicator predominates over the brownish colour of the mesenteric secretions and can be identified through the wall of the exposed digestive tract. A pH-meter. A glass electrode was immersed in 6 ml of intestinal contents which were removed quickly from the dissected intestine of larvae. From 1 to 1.8 ml haemolymph was collected with a syringe introduced in each larva above the 6th abdominal spiracle. The values were calculated from the averages of 5 or 6 pooled samples. Study of the redox potential. A P50 TACUSSEL potentiometer with a platinium electrode and a fixed was used. The reference electrode calomel measurements were made by quickly immersing the electrodes in the contents of the mid- or hindgut of quickly-dissected and longitudinally-opened intestines.

AND METHODS

Materials Oryctes nasicornis (L.) larvae were bred in the ‘Laboratoire de Butte Biologique de La Mini&e (I.N.R.A., Versailles) on a fermenting mixture of wood and dung according to the method of HURPIN and FRE~NEAU(1964). The third and last larval instar was used in this study. The larvae were reared at 28°C (the optimal temperature for their development) on two different media: dung-wood mixture, which is similar

Study of the enz+vmeactivities Micro-method. Qualitative 819

and

semi-quantitative

820

COLETTE BAYON

evaluation of enzyme activities was carried out with an lyophilised and taken up in l/3 of its original volume ‘API ZYM’ (API-System) using the method of of distilled water. The concentrated solution was NARDON and PLANTEVIN(1970). The method was deproteinised with 1 ml N perchloric acid, the applied to the intestinal epithelia, the haemolymph precipitate discarded and the sugars separated by thin and the gut contents (0.1 g of sample in 1 ml of doublelayer chromatography (TLC) using ADACHI’S distilled water). technique (1965). Migration was carried out on Carboxymethylcellul activity. The technique Kieselgel6O”F 254 (Merck) concentration zone plates employed was based on that of POTTS and HEWITT impregnated with 0.1 M sodium bisulphite and (1973), the substrate being a solution of activated at 120°C for 1 hr. The solvent was a carboxymethylcellulose (BDH Chemicals propanol-water (85:15) mixture and the spots Ltd., sodium salt, degree of substitution: 0.7-0.8): 1 gin 100 developed with a mixture of 0.5 g thymol, 5 ml H,SO, ml of 0.02 M phosphate buffer at pH 6.9. The samples and 95 ml ethanol. Colouration appears after heating analysed were: intestinal walls ground in a Potter the plates to 110°C for 15 min. The standards used apparatus with 0.08 M citrate-phosphate buffer at pH were 1 g/l. solutions of glucose, trehalose, xylose and 5.8 (3 g in 4 ml buffer) and intestinal contents in buffer cellobiose. at pH 10.4 for the mesenteron and pH 7.8 for the Volatile fatty acids ( VFA). The tests were carried proctodeal dilation (1 g contents in 1 ml buffer). The out on 25 gpools of samples stored at - 15°C acidified samples were used either unmodified or centrifuged at with 5 ml saturated tartaric acid and degassed for a few 10,000 rev/min, in which case the supernatant was seconds under vacuum in order to eliminate the CO, analysed. which is present in the intestinal contents whatever On each sample 2 identifical assays (A) and 2 their origin. Vapour distillation was carried out at constant volume and temperature according to controls (C,and C,) were carried out. For the assays JAULME’Smethod (1951). The distillate (200 ml) was 2 ml homogenate were mixed with 1 ml substrate and collected and immediately titrated against 0.1 M or incubated for 1 hr at 37°C. Control C, was composed 0.01 M NaOH with phenolphthalein indicator. of 2 ml homogenate and 1 ml substrate incubated at The titrated distillates were neutralized with excess 4°C and for control C,, 2 ml homogenate and 1 ml of buffer was incubated at 37°C for 1 hr. Furthermore NaOH and evaporated under vacuum then under nitrogen. The remaining 20 ~1 deposit was taken up in this experimental series (A, C,, C,) was repeated on 0.2 ml 20% formic acid and analysed by gas-liquid the gut contents in anaerobic conditions for which a layer of liquid paraffin was placed over the incubation chromatography (GLC). The GLCs employed were a Perkin-Elmer 880 and a Girde175. GS. PT with flame medium. ionisation. The carrier gas was nitrogen (20 ml/min) Carboxymethylcellulase activity is detected by the saturated with formic acid (by bubbling). Formic acid presence of reducing sugars and glucose formed on does not give a signal in flame ionisation so that it is a hydrolysis of the substrate. Colourimetric methods substance which cannot be detected using this were tried for the assays but both the controls and the technique. A 3 m long, l/8 in. diameter stainless steel samples to be tested presented a strong brown column was used packed with Chromosorb 60-80 colouration which is due, at least partly, to the colour mesh HMDS impregnated with 10% 20 M Carbowax. of the mesenteric secretions. It was observed that the interfering substance absorbed at all visible uv The initial temperature was 140°C and the final temperature 190°C; it was programmed to rise at wavelengths. No deproteinization technique was able 3”C/min. The samples were injected in quantities of 0.2 to completely eliminate the discolouration. Micro~1.The retention times of the separated substances were assay methods were therefore used on the samples compared with commercial VFA standards. Valerie which had been diluted and then deproteinised. The acid was used as an internal standard for quantitative results obtained give only an indication of the presence analysis. The results are expressed in equivalents of of glucose or of reducing sugars by comparison acetic acid. between the test samples and the controls. The Methane detection. Methane was assayed in the quantities of sugar measured in the controls should intestinal contents by rapidly dissecting out a section not be taken as being the actual quantities of sugar of intestine and placing it in a 15 ml vial which was originally present in the samples. The reducing sugars quickly sealed with a ‘rubber stopper. A needle was were assayed using Somogyi’s reagent according to the introduced to open the section and break up the method of NELSON(1944) after deproteinization with contents and, after a period of stabilization of the sodium tungstate. The level of glucose was determined enclosed gasses, 2 ml of the atmosphere was removed enzymatically after deproteinization with uranyl and analysed by gas chromatography. Thus it is the acetate (G.O.D. method from Bijhringer or Merck). quantity of methane liberated by each intestinal The percentages of light transmitted and the optical segment which was evaluated (the amount of CH, densities were measured with a PM 2 DL Zeiss dissolved in the gut contents was not considered). spectrophotometer. The results are given on a Larval methane release was investigated by placing a calibration curve obtained with a range of glucose larva in a sealed 15 ml container for 1 hr at 28°C and standards. sampling 2 ml of the atmosphere as above. Substances formed during gut transit Qualitative and quantitative methane assay was This study was carried out on the intestinal contents carried out by gas chromatography under the at various points along the digestive tract, on the food, following conditions: the GLC was a 88 1 type Perkinon the faecal pellets and also on the haemolymph. Elmer equipped with a flame ionisation detector. The Disaccharides and monosaccharides. Samples injector temperature was 250°C and that of the oven weighing 10 g were centrifuged. The supernatant was 25°C. A stainless steel column (interior diameter l/S,

Anaerobic carbohydrate fermentation in Oryctes nasicornis

length 2.5 m) was packed with Porapak Q.80-100 mesh. The carrier was nitrogen at 25 ml/min. The nature of the detected gas was checked on a mass consisting of a Girdel 3000 spectograph chromatograph fitted out with the column described above and coupled to a M.A.T. Varian CH, type mass spectrometer through a separator of the kind proposed by WATSON and BIEMANN (1965). The vector, helium, flowed at 20 ml/min. Ionisation was electronic (70 eV electrons). The temperature of the source was maintained at 230°C. The retention times of the gas to be characterized were compared with those of methane and found to be identical. The quantities of methane injected were calculated from a calibration curve (peak height/quantity of methane injected) based on methane-air standard mixtures.

RESULTS Physico-chemical conditions of the intestinal contents Redox potential study(Table 1) In Oryctes larvae only the mid- and hind-gut is developed fully and only here is food found continuously (BAYON, 1980). Whatever the food ingested the contents of the midgut have a slightly positive redox potential of the order of + 30 mV, whereas that of the contents of the proctodeal dilation is clearly negative, of the order of -80 mV even reaching -100 mV when the foodstuff is composed of ligno-cellulose material. So, overall, the gut contents have a reducing capacity which is not noticeably changed on a pure a-cellulose diet. The reducing power becomes greater in the hind-gut. pHstud_v (Table 2, Fig. 1). The pH ofthe wood-dung foodstuff and that of the feacal pellets are near neutral

821

Table 1. Electrometric measurements of the redox potential (in mV) of the mesenteric and proctodeal contents with two different foodstuffs Food: dung-wood

Midgut Mean + 27(+18)

Food: pure r-cellulose

Proctodeal dilation

Midgut

- 83(+22)+

37(+_ 16)- 50(+_ 17)

Each mean corresponds from 6 insects.

Proctodeal dilation

to the measurements obtained

The mid-gut contents are characterized by high alkalinity values: 12.2 was recorded in the Mi,

portion which reaches from the second group of caeca to the proctodeal valve. Although the proctodeal contents showed a gradient of decreasing pH values they remained above 7. The intestinal contents of Oryctes larvae are therefore strongly alkaline in the mid-gut and weakly so in the proctodeal dilation. Similar results were obtained in insects fed on pure c( -cellulose. The use of coloured pH indicators confirms the electrometric results and gives further information on the localization of the pH modifications. The colour change of thymol blue, cresol red and phenol red show the abrupt increase in the pH occurring in the fore-gut from the reflux of mesenteric secretions in the stomodeal lumen at the time ingestion. The colour change of thymol blue, cresol red, phenol red, neutral red and bromothymol blue demonstrates the progressive pH decrease in the hind-gut. This decrease starts at the proctodeal valve where the 4 Malpighian tubules join the intestine and continues in the

Fig 1. pH of intestinal contents. The scheme shows the digestive tract. AI: anterior intestine; aMi,: anterior midgut 2; AP: anterior proctodeum; aPD: first part of proctodeal dilation; C,, C,, C,: caeca 1,2, 3; DW: aliment (dung-wood); FP: faecal pellet; Mi,: first part of midgut; Mi,: second part of midgut; MT: Malpighi vessel; PD: proctodeal dilation; pL: posterior lobes; pMi,: posterior Mi,; PDA: proctodeal dilation ampulla; pPD: second part of proctodeal dilation: R: rectal chamber. Electrometric measurements: DW-Mi,-aMi,-pMi,-AP-FP. Dye colour measurements: AI-pL-aPD-pPD-PDA-R.

..

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results (mean f S.E.M.)

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7.28

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between 8.2

between 1.2

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Food

coloured indicators

Results with

Methods

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976

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and 8.8

between 8.2

Mi,

Table 2. pH values in samples

(Mi,), proctodeal

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ampulla

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part near the wall

Proctodeal

and by electrometric

Anterior in the contents

indicators

(Mi,), second part of the midgut

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7.10

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Anaerobic carbohydrate fermentation in 0ryr1r.c nasicornis

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824

COLETTEBAYON

proctodeal dilation and the rectum. Pure a-cellulose fed animals show similar phenomena. In the anterior half of the proctodeal dilation, the contents which are in contact with the epithelium have a slightly lower pH. This observation can be made on insects on a phenol red impregnated diet at the instant of digestive tube dissection. The peristaltic contractions induced by the shock of the operation cause the differences in colouration to disappear quickly.

esterases and they possess no valine-cystineaminopeptidase and no protease activities. The zymograms of the haemolymph gave results similar to those found by NARDONand PLANTEVIN (1970). Carboxymethylcellulase activities

Enzyme activity investigation (Table 3) The ‘API system’ allowed 19 enzyme activities to be tested. This analysis underlines certain characteristics which are commonly found in insects. It is the mesenteron which presents the greatest number of enzyme activities. The generalized presence of alkaline and acid phosphatase, esterase, lipase, aminopeptidase and glycosidase activities are linked to the mesenteric epithelium. The relatively high number of enzyme activities fomld in the short stomodeum and in the proctodeum are due, in part, to the fact that the method used detects enzymes of general cell metabolismwell. It isuseful to point them out since their presence does not imply that they are involved in digestive processes: this is the case for phosphatase, esterase and amino-peptidase, as for fi-glucosaminidase which is known to be widespread in all species. The latter can be involved in the formation of cuticle layers and its presence is not surprising. Thus the zymograms to be studied should be those which, in the light of present understanding, appear to be original. There is a high alkaline phosphatase activity found not only in the mid-gut but also in the proctodeal dilation and the rectum. This enzyme is involved in transfer phenomena and the hind-gut of Oryctes nasicornis would seem to be the site of absorption phenomena, both the rectum as in all insects (PHILLIPS, 19771, and the proctodeal dilation. The mid-gut is characterized by strong esterase activity (mainly C8”). It can also be seen that the mesenteron shows strong esterase, lipase, leucine- and valine-aminopeptidase activities. The Mi, region shows trypsin activity. The Mi, region shows chymotrypsin activity. Positive reactions are more numerous in the hind-gut than in the fore-gut in contrast to the findings in other insects (PLANTEVIN and NARDON,1972). Note should be taken of the high P-galactosidase activity of the proctodeum. The liquid fraction of the gut contents is not rich in glycosidases; only rectal contents display several glycosidase activities. The proctodeal contents are not rich in

The intestinal epithelia. The experiment was repeated 10 times and the t test was applied to the weights of reducing sugars (or glucose) found in the assays and the blanks. For the same segment, these values are not statistically different. The technique used does not provide evidence for the existence of an enzymatic system, adapted to the lysis of carboxymethylcellulose, secreted by the intestinal epithelia of Oryctes larvae. The intestinal contents. The experiment was repeated 8 times and application of the f test shows that the assays and the controls are not significantly different. The technique used does not provide evidence for the existence of carboxymethylcellulase activity in the intestinal contents in vitro. Mono- and di-saccharides Neither cellobiose nor glucose are identified in the food or in the different parts of the intestinal contents of animals fed the wood-dung mixture or pure cc-cellulose. However a pentose (xylose) does appear in the mid-gut of wood-dung fed animals but not in animals fed cc-cellulose. Volatile fatty acids (Tables 4 and 5) Study of wood-dung fed animals. The wood-dung medium which was used as foodstuffs contained little VFA: 0.3 x 10m6 M g-l. However the level of VFA present in the mesenteric contents reached 25 x 10m6 M g-l. The proctodeal dilation only contained 7.2 x 10-6Mg-1.Therectumhadnomorethan1.8x10-6 M g-l. The haemolymph had 1 x 10m6 M VFA g-l. The low variation of the VFA levels in each segment shows that the values are constant for a group of larvae of the same age. As the gut contents pass through the mesenteron the level of VFA of the lignocellulose material is increased 83-fold. In the proctodeum however the level of VFA falls and becomes only 24 times greater in the proctodeal dilation and 3 times in the rectum. The intestinal contents possibly and the mid-gut certainly are therefore the site of VFA formation. Our investigation does not consider the yield of this reaction but shows that the level of VFA is constant for a given segment

Table 4. Amount of total VFA expressed in acetic acid equivalents. Two different values according to the foodstuffs: wood-dung (A) and pure a-cellulose (B)

Food (1 g)

Samples Diet

A

B

0.3 Total acidity (10e6 moles per g fresh wt) (mean f S.E.M.)

Titration Chromatography

0 071 0.2 i 0.09

0

Proctodeal dilation (1.967 g) A B

Faccal pellets (0.06 g) A B

22.2

7.2

7.7

1.8

0.9

3: 21.1

1’3 6:8

277

I?2

0; 6.9 * 1.0

oz9 1.0 * 0.08

O?l 1.0 + 0.08

Midgut (1.732 g) A B 25.0 + 1.9 23.9 + 1.4

Haemolymph (1 g) A B 1.0 L 0.9 1.5 zk 0.9

1.48 0:‘8 1.5 OF7

825

Anaerobic carbohydrate fermentation in Oryctes nasicornis Table 5. Percentages of different VFA according to foodstuffs

_

Insects fed with dung-wood

S, Acids

Midgut

Acetic Propionic Isobutryric Butyric lsovaleric

96 2 0.8 0.2 1

Insects fed with pure a-cellulose

Proctodeal dilation

Faecal pellets

Haemolymph

Midgut

90 7

95 4 0.5 0.5

94 5 0.5 0.5

91 9 -

I

1 1

and that there is a falling gradient along the digestive tract. 5 VFAs are found to be present in the mid-gut and proctodeal dilation contents: acetic, propionic, isobutyric, butyric and iso-valeric acids. 4 VFAs are found in the foodstuff, in the rectum contents and in the haemolymph: acetic, propionic, butyric and isovaleric. The total quantities of VFA detected by GLC confirm the results obtained by titration. The percentages of the various VFAs present are given in Table 5. Acetic acid is by far the most abundant, constituting 907, of the total. The propionic acid content varies from 2 to 7%. The other acids were present in such low quantities that they should be considered as traces. The traces of butyric and isovaleric acids were present in slightly greater quantities in the faecal pellets than in the intestinal contents. Study of the animals fed pure a-cellulose. VFAs are present throughout the intestinal contents and in the haemolymph. A falling gradient exists for the level of VFAs along the intestine. The quantities of VFA present are not significantly different to those found in wood-dung fed animals. Only acetic and propionic acids are found to be present in the gut contents and traces of butyric acid are however found in the faeces. Acetic acid accounts for 90% of the VFA’s and is the only one detected in the haemolymph. Methane study

The chromatograms corresponding to the mesenteric contents are little different from those obtained by injecting the ambient laboratory air. Methane is not detected in the mig-gut contents of either dung-wood fed or cr-cellulose fed animals. However chromatograms corresponding to proctodeal dilation contents all present a large peak identified as methane. The proctodeal dilation contents liberate on average 53.9 k 3.5 nmoles methane in wood-dung fed animals and 45.5 _t 3.7 nmoles methane in cc-cellulose fed animals. The t test applied to both groups of samples showed them to be not significantly different. The yield per gram of gut contents was 22.4 f 4.58 nmoles for wood-dung fed animals and 20.7 i 1.3 nmoles for cc-cellulose fed animals. The yield per gram of the gut contents is therefore slightly lower in cc-cellulose fed larvae. Methane release per animal was studied over 1 hr periods. For animals fed wood-dung mixture release of 308-372 nmoles was observed in 1 hr. The hourly release on a per gram basis of the proctodeal contents was therefore 128-155 nmoles, i.e. 34-41 nmoles per gram of the whole animal. The a-cellulose fed larvae liberated 343-380 nmoles hr-l, i.e. 156-173 nmoles

Proctodeal dilation

Faecal pellets

Haemolymph

90 10 -

88 6 6 -

100

-

hr - l g - r proctodeal dilation contents or 38-42 nmoles per g of the whole animal.

DISCUSSION Cellulolysis Oryctes nasicornis larvae feed solely on decomposing sawdust, and it has been proposed that the hydrolysis products of cellulose constitute an energy input for the insect. The hydrolysis of cellulose to cellobiose requires the synergistic then successive action of two types ofexocellulases: C, and ~~(SELBY, 1968). The Cx enzymes act on carboxymethylcellulose (ALMIN and ERIKSSON, 1968). No carboxymethylcellulase activity was identified in the digestive tract of 0. nasicornis. These results confirm those of PIAVAUX and DESIERE (1974) and of MARCUZ~I and TURCHETTO-LAFISCA(1977) on Oryctes and of RIPPER (1930) and SCHLOTTKE (1945) on other Scarabaeidae. However Melolontha (RICOU, 1958) and Sericesthis (Soo Hoo and DUD&SKI, 1967), have a specialized enzyme system mainly localized in the proctodeum. In Sericesthis the authors do not exclude the possibility that the enzymes involved are of bacterial origin. The high alkalinity of the mesenteric contents is not compatible with the cellulase optimum pH values which are of the order of 5.6-6.1 (MANSOUR and MANSOUR-BECK,1934; LASKERand GIESE, 1956). Furthermore in Oryctes, as in Geotrupes and Seriscesthis, the results indicate the presence of /I-glucosidases in the gut walls. Soluble sugar analysis of the gut contents of larvae fed on dung-wood or a-cellulose do not show the presence of cellobiose, which would result from the activity of C, and C, enzymes, or of glycose which would be formed by the /I-glucosidases. The formation of various volatile substances is demonstrated. The results are comparable in larvae fed on the wood-dung mixture and in those fed on a-cellulose. The presence of CO, was observed, but was not quantitatively investigated. VFAs were found to be present throughout the intestinal contents and in the haemolymph; acetic acid is by far the most abundant (over 90%). Each intestinal segment was seen to present a constant VFA concentration: 0.15% in the mesenteron, 0.04% in the proctodeal dilation and O.Ol’A in the rectum. The gradient shows that VFAs begin formation in the mid-gut, but it is not incompatible with the hypothesis that the formation continues in the proctodeal dilation, but this is not a detailed study of the yield of VFA formation. The gradient which appears is the result of opposing

826

COLETTE BAYON

processes-that of formation and that of disappearance. Indeed the passage of VFAs through the intestinal wall should be considered since these substances are present in the haemolymph and again it is acetic acid which predominates. Methane is detected but only in the proctodeal dilation and methane release by the larvae was determined to be 308-372 nmoles hr - l. So VFAs, CO, and CH, are formed in the intestine of Oryctes: they are also substances known to be derived from the fermentation of hexoses in certain herbivores (HUNGATE, 1966; MURRAYet al., 1976). This very specialized form of cellulose digestion has been studied in the cow: the ingested fodder is lysed and fermented by micro-organisms and the VFAs formed are oxidized in the tissues of the host. A mutually beneficial relationship is established therefore between the ruminant and the ciliates and bacterial populations of the rumen. These processes are not restricted to ruminants since a similar situation is found in the intestinal caecum of Lagomorpha (PARKER,1976), in the pony, in the stomach of certain apes and marsupials (HUNGATE,1966) and even more recently in the green turtle Chelonia mydas (BJORNDALL,1979). In insects it was the lower termites which were first studied. HUNGATEproposed that the flagellated microfauna of the termites ferment polysaccharides in the proctodeal dilation of these insects. So a functional convergence between the digestive physiology of two very different zoological groups is demonstrated. The works of HUNGATE (1936, 1938, 1939) concern Zootermopsis. Other authors confirmed such hypothesis in Reticuiitermes and Coptotermes (BREZNAK, 1975; THAYER, 1978). Breznak also showed similar occurrences in Cryptocercus punctulatus. Comparable results allowed this theory to encompass the higher termites which possess a population of bacteria in their proctodeum: POCHONet al. (1959) in Sphaerothorax, KOVOOR (1967) in Microcerotermes and FRENCH (1975) in Nasutitermes. The redox potential conditions in which sugar fermentation occurs in Oryctes were investigated. The potential differences of the mid-gut contents were slightly positive whilst those of the proctodeum were clearly negative. The study of biodegradation processes in the soil has allowed characteristic stages to be distinguished through the redox potential values (PAAR. 1969). Values of less than + 300 mV indicate reducing conditions in which facultative anaerobiosis can exist: degradation of organic matter accelerates and soluble products can accumulate. For negative values, reducing conditions intensify and conditions of strict anaerobiosis prevail: from - 100 mV carbohydrates are reduced completely and methane can be generated. In general the intestinal contents of Oryctes are reducing, becoming more so from the midto the hind-gut as shown by VEIVERSet al. (1980) who studied termites. Fermentation occurs anaerobically, the two intestinal segments studied presenting different degrees of anaerobiosis: facultative in the mesenteron and obligatory in the proctod+m. In the hind-gut the reducing conditions are compatible with the formation of methane. The redox potential in the stomach of ruminants reaches values of the order of -300 mV and the methane yield is 20 times higher (BAYONand ETIEVANT,1980).

The steps involved in polysaccharide digestion

Intestinal transit studies show that the fore-gut and the portions of proctodeum not including the proctodeal dilation and the rectum are only simple tubes regulating the transport and are normally empty, that the coefficient of the digestibility is 570; (BAYON,1980). Only the mid-gut and the proctodeal dilation were studied, each being characterized by the constant presence of regularly renewed alimentary bolus of constant volume and no noticeable stasis. T/le mesenteric stage. The mesentery transit time at 28°C is 8+ hr and the contents are not churned during this time. Throughout the segment, the ligno-cellulose fragments are impregnated with mesenteric secretions and the density of the bacterial population is doubled (BAYON, unpublished observations). Reducing conditions form an anaerobic state which is favourable to the decomposition of organic material. The pH is strongly basic with an increasing gradient from the anterior sphyncter to the proctodeal sphyncter. Alkalinity can reach pH 12 in the Mi, region. Such values are exceptional in insects where a pH greater than 9.5 is rarely observed . Lepidoptera caterpillars present the highest pH values: e.g. 10.3 in Bombyx mori (WATERHOUSE,1949). However it is impossible to make any correlation with a plant-based diet, since LINDERTROM-LANG and DUSPIVA (1935) reported keratin degradation by protease active in a reducing medium at pH values greater than 10. In Scarabaeidae larvae, high pH values are always found (WERNER, 1926; SCHLOTTKE,1945; R&SLER, 196 1). Zymogram studies demonstrate the existence of protease activity as already demonstrated by SCHLOTTKE (1945) in Cetonia, Potosia and Dorcus, by RICOU (1958) in Melolontha, by SOO Hoo and DUDZI&KI (1967) in Sericesthis. Thus it is thought that the mesenteron of Scarabaeidae larvae is involved in protein metabolism. In current research into the processes involved in the biodegradation of lignocellulose materials a ‘pre-cellulolytic’ phase is described during which the lignins and hemi-celluloses constituting the plant cell walls, although not lysed, are at least modified, rendering the celluloses accessible to the synergic action of cellulases (KIRK, 1971). Among the ‘precellulolytic’ agents are chemical factors like basic media (HUNGATE,1966; BAILEYet al., 1968). The mesenteron of Oryctes creates the chemical conditions for the ‘precellulolysis’. The contents are characterized by the absence of glucose and cellobiose, but the presence of high quantities of VFAs: 2.598 mg in one mid-gut. The great constancy of the levels is not altered when the diet is pure cr-cellulose. So celluloses are fermented in anaerobic conditions until the stage of VFA formation. It is possible that hydrolysis can only be performed on cellulose molecules in which degradation has begun before the foodstuffs are ingested. Classically the mesenteron of insects is considered as being an absorbing intestinal segment. VFAs are present in the haemolymph where acetic acid is again preponderant. The constant levels of VFA in the intestine might be maintained due to an excess of VFAs formed in relation to the quantity absorbed by the haemolymph. In Popillia haemolymph acetic acid has also been detected (STUBBLEFIELD et al., 1966).

Anaerobic

carbohydrate

fermentation

The proctodeal stage. The proctodeal dilation contains numerous bacteria and parietal flora (BAYON, unpublished.). The epithelium presents an ultrastructure which can be related to the absorption processes (NOIROT and BAYON, 1969; BAYON, 1971). The proctodeal dilation resembles anatomically and cytologically the paunch of termites (NOIROT and NOIROT-TIMOTHEE,1969) and has the characteristics of the bacterial segment described by MARTOJA (1966). The contents have a constant volume and are continually renewed over 6 hr. The pH is maintained at slightly basic values. The sharp drop in the pH before the entry into the proctodeal dilation is caused by the secretion of the 4 Malpighian tubules, but the falling pH gradient in the proctodeal contents is due to the fermentation which becomes progressively more intensified and the reaction products which tend to neutralize the medium. The reducing conditions of strict anaerobiosis favour fermentation: the degradation of organic matter accelerates and the reduction process is continued to the stage where methane is formed, later to be completely released with the faecal pellets. The VFAs produced do, however, remain present in small quantities: 0.849 mg in one proctodeal dilation. Neither glucose nor cellobiose are present in the contents. The hypothesis that the VFAs diffuse across the proctodeal wall into the haemolymph is not contradicted since present knowledge of the proctodeal cuticle of insects indicates that they are permeable to this class of molecules (PHILLIPS, 1977). In the proctodeal dilation two processes contribute to keep down the level of VFAs: diffusion across the intestinal wall and methanogenesis. Thus the proctodeal dilation of Oryctes can be anatomically and physiologically compared with the paunch of higher termites. It shows also functional convergence with the stomach of ruminant mammals possessing the various characteristics defined by HUNGATE: alkaline action precedes its activity; the contents are continually renewed; anaerobiosis is obligatory; there is absence of a host cellulase; microorganisms are present; there is no detectable glucose formation. VFAs are present at low levels and methane is formed.

Acknor~led~en~ents-I am grateful to Dr. HURPIN B., Director of Station de Lutte biologique de La Mini&e and P. ROBERT(INRA-Versailles) for providing Orycfes. I also thank P. DUBOISand J. RIGAUD of Station Arames (INRADijon) for providing the technique for analysis VFA, Dr. TOUTAIN (Laboratoire de PBdologie-Nancy) for the opportunity to use the potentiometer, and to Dr. AU~ISSIER N. (Biochimie cellulaire-Dijon) for helpful discussions.

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in Orycfes

nasicornis

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