Proceedmqe of the 7th Internatumal WorkLng Conference on Stored-product Protection - Volume 2

truncatus ( Horn)Bacterial presence in Prostephanus(Coleoptera: Bostrichidae)

M. Vazquez-ansta", G. L. Basurto-cadena", R. Vargas-becera' and R. E. HmOlosa-rebolla?

Abstract

Bactena from the digestive system of larvae, adults, anddead adults of Prostephamus truncatus (Horn), maizestored pest, from MeXICO, Tanzama, and Togo wereIsolated and Identified Pseudomonas [iuorescene ,cellulolytic bactenum, was Isolated from the guts of thethree colomes of P. truncatus and the two insect stagesstudied. The presence of this bactenum suggests that couldbe involved m the P truncatus digestive system,therefore this msect is able to survrve in a great vanety ofenvironments and to be considered as a potential pest for anyagncultural crop Streptococcus faecucm , Streptococcusdysgalactiae, and Burkholderui cepacui , Isolated fromguts and dead insects, could be the cause of P truncatusmortah ty dunng stress penods

Introduction

The large gram borer, Prostephanus iruncatus (Horn),has been recogmzed as a common but mmor pest of farm-stored maize m MeXICO and Central and South Amenca.Tigar et al (1994) reported ItS distribution and abundancem MeXICO. There are also pubhshed records of ItSoccurrence in the Amencas (Rees et al ,1990) Dunng theearly 1980s, this insect was introduced into Africa and ISnow the most destructive pest to farm-stored maize (Zeanwys L ) and dned cassava (Manzhot esculenta Crantz )and wooden structures (Hodges, 1986; McFarlane, 1988 )Cellulose, the majOr component of most hgnocellulases, IS

a'lmear homopolymer of 13-1,4-hnked glucose EnzymatichydrolYSIS of cellulose to glucose occurs by the action ofcellulase a mIxture of enzymes WIth dIfferent actiVIties andspeCIfICIties (Breznak and Brune, 1994)

1 Centro de Investlgaclty de Estudlos Avanzados del IPN( CINVEST AV-IPN ), Departamento de Blotecnolog y BloqUlca,Ullidad de Blotecnolo ge Ingellier Gellica de Plantas

2 Instltuto de ClenCias Agrolas, Departamento de Ahmentos,Ulliversidad de GuanaJuato, MlCO

3 Departamento de MicrobIOlogy , Escuela Naclonal de ClenclasBlohcas del Instltuto POhtlllco Naclonal, Mlco

In white rot fungi and probably m insects, cellulose ISdigested by extracellular enzymes to glucose that can be usedto meet the carbon and energy requirements of an orgamsm(Martin, 1991)The functions of endosymbiotic rmcroorgarusms in beetles

are to synthesize some nutntionally essential substances thatare lacking m their diet or to make possible the use of somecomponents of the food that beetles would be unable todigest (Crowson, 1981).Many wood-eatmg insects use cellulolytic enzymes that

are erther endogenously secreted or that are produced bysymbiotic rrucroorgantsms such as protozoa, bactena, andfungi (Becker, 1977; Breznak, 1982; Orpm and Anderson1988 ; Coughlan, 1992) Other wood or fohage-ea tmginsects are completely noncellulolytic and denve no benefitfrom the cellulose consumed m their diets: instead, theyrely on the degradation of other polysacchandes such asstarch for their nutrition (Martin, 1983)The presence of cellulolytic bactena m adults and larval

guts of P iruncatus from Tanzama suggests that tlusmsect could be able to digest cellulose (Vazquez-Ansta etal , 1997)

Materials and Methods

Insects

Three colomes of P trumcatus , from MeXICO,Tanzama, and Togo, were mamtamed as previouslydescnbed (Vazquez-Ansta et ai, 1997). Fifteen P.truncatus last mstars OOd old) and 2-wk-old adults weredissected by usmg a cold drop of Ringer solution

Gut extract preparation

Fat tissue was phYSIcally removed. Whole guts werethoroughly nnsed 3 times WIth cold, stenle dIstilled waterand then homogemzed by usmg a stenle Potter homogemzermImi of stenle, dIstIlled water Rmses and homogemzatIonwere done m a bIOlogIcal safety cabmet.

Dead insects extract preparation

Twenty five dead msect adults of each colony were used.Dead msects were nnsed 3 times With stenle distilled water,then were washed dunng 15 mm With 10% chlondesolution, and fmally were thoroughly nnsed 3 times With

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Prcceeduiqe of the 7 th Internatwnal Working Conference on Stored-product Protection - Yoiume 2

sterile distilled water. Dead insects were homogemzed byusing a stenle Potter homogemzer m Lrnl of stenle distilledwater. Dead insects extract preparation was done in abiologrcal safety cabinet (Rady, et al. , 1992).

Isolation of aerobic microorganisms

A loopful of guts and dead msects homogenates wasinoculated in petri plates contammg nutntive agar (23g/hter ) This procedure was repeated to obtain theseparation of rmcrobial colomes. Pure cultures wereobtained from the Isolated colomes by inoculation in nutrientagar (23 g/Irter): These pure cultures were used for furtherassays. Microorgarusms were conserved as stock cultures bymaculation of a pure culture loopful in test tubes withmclmed nutnent agar (23 glIiter). The streak-plate methodwas used for inoculation. Petn plates and test tubes wereincubated at 29 ± I°C under aerobic condttions (Brock et al.1997a)

Gram staining and bacterial identification

The gram-stammg and identifrcation techmques wereperformed as previously descnbed (Vazquez-Anst 1997).

Results

Isolation of aerobic microorganisms

Seventeen bacteria were Isolated from the guts of P.truncaius SIX were from the Mexican colony and werenamed Ml and M2 (from adult guts), M3 and M4 (fromlarval guts), and M5 and M6 (from dead adults) SIX werefrom the Tanzanian colony, 21 and 22 (from adult guts), 23and Z4 (from larval guts), and 25 and 26 (from deadadults) The last 5 were from the Togo colony, Gl and G2(from adult guts), G3 and G4 (from larval guts), and G5(from dead adults) .

Gram staining

EIght gram-negative (M2, M4, 21, 23, 26, Gl, G4, andG5) and 9 gram-positive (Ml, M3, M5, M6, 22, 24, 25,G2, and G3) bactena were Isolated

Bacterial identification

Table 1 shows the identmcation of the bactena Isolatedfrom the P truncatus adults and larval guts, as well asdead msects

Discussion

ThIS research shows the presence of Pseudomonasfiuorescens, cellulolytic bactena; Streptococcus(Enterococcus) faeclum; and Streptococcus dysgalactiaem the dIgestive system of P. truncatus from MexIco,Tanzama, and Togo; m addItion to Burkholderw cepacia

(formerly known as Pseudomonos -cepacui ) from deadadults.

Table 1. Bactena Isolated and identified from P.Truncatus

Ongm of Source CodeP. truncatus

Bactena

MeXICO

adult guts Ml Streptococcus faeciurn

adult guts M2 Pseudomonas fluorescens

larval guts M3 Strepiccoccus dysgalactme

larval guts M4 Pseudomonas fl uoreecens

dead msects M5 Streptococcus [aecucm.

dead msects M6 Streptococcus [aecium

adult guts 21 Pseudomonas fl uorescens

adult guts 22 Streptococcus faecium

larval guts 23 Pseudomonas fluorescens

larval guts Z4 Streptococcus faeciurn

dead msects 25 Streptococcus [aeciurn.

dead msects 26 Burkholderui cepacui

adult guts Gl Pseudomonas fluorescens

adult guts G2 Streptococcus dysgalactwe

larval guts G3 Streptococcus dysgalactwe

larval guts G4 Pseudomonos fluorescens

dead msects G5 Burkholderui cepacia

Tanzama

Togo

The use of nutntrve agar in the isolation of these bactena,instead carboxymethyl cellulose (Vazquez-Ansta et al ,1997), gave umform results among the adults and larvalstages.The bactenum, P. [luorescens , causes food spoilage in a

WIde vanety of agncultural products, mcludmg milk, meat,and fresh vegetables. _Jt !~_,rv~p.ollS1ble for a substantialproportion of postharvest losses of agncultural commodities(Liao and McCallus, 1997), and rarely considered as anammal pathogen (Brock et aI., 1997b). Hence, theisolation and idennfrcauon of these bactena from the adultsand larval guts of beetles from 3 P. truncaius colomes ofdifferent origin, could confirm that tlus insect has the abilityto feed on dIfferent types of carbohydrates; that IS, P.truncatus does not depend on food where starch IS the mainsource of carbohydrates such as maIze and cassava, and Itmay successfully establIsh as a pest m a large vanety ofcropsOn the other hand, the genus Streptococcus contams a

WIde vanety of speCIes WIth qUIte dIstmct habIts, whoseactiVIties are of conSIderable practical Importance tohumans. The genus Enterococcus mcludes streptococci that

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Proceedings of the 7th International Workmg Conference on Stored-product Protection. - Yolume 2

are pnmanly of fecal ongm and are considered pathogenicfor humans (Klare et al., 1995), with some strams ofconsiderable antibionc resistance (Chadwick et al., 1996;Henmng et al , 1996; Mato et al , 1996). Then, as S.[aecucm. is a human pathogen, It could be considered as anopportunistic pathogen of P. truncatus too, and can beresponsible of its death during starvation periods. The sameconsideration should be made With the presence ofBurkholderia cepacia , isolated from dead insects, becauseIt IS now demonstrated that this bactenum IS anopportunistic pathogen m patients WIth fibrocystic lungdisease, due to Its mcreasmg association WIth fatalpulmonary infections (Reboh et al., 1996; Revets et al. ,1996).The presence of S. dysgalactiae m the digestive system

of P. truncatus seems to have no connection whit anydigestive process, because It ISknown that tlus bactenum ISmvolved m the mastitis of dairy cattle, which IS anmflammation of the bovine mammary gland (SImpson,1995).Because of the urnformrty of the bactenal presence among

the three colomes of P truncatus, make difficult toestablish that they have different ongm, as It was suggestedby Vazquez-Ansta et al. (1997).

References

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