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Digestion of Starch Granules from Maize, Potato and Wheat by Larvae of the
the Yellow Mealworm, Tenebrio molitor and the Mexican Bean Weevil, Zabrotes
subfasciatus
Author(s): Elaine A. Meireles Cntia N. B. Carneiro, Renato A. DaMatta, Richard I. Samuels and Carlos
P. Silva
Source: Journal of Insect Science, 9(43):1-8. 2009.
Published By: University of Wisconsin Library
DOI: http://dx.doi.org/10.1673/031.009.4301
URL: http://www.bioone.org/doi/full/10.1673/031.009.4301
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Digestion of starch granules from maize, potato and wheat by
larvae of the the yellow mealworm, Tenebrio molitor and the
Meican bean weevil, Zabrotes subfasciatus
Llalne A. Melreles
1,
|, Clntla N. 8. Carnelro
1,a
, Penato A. DaMatta
2,b
, Plcbaro |. Samuels
3,c
ano
Carlos P. Sllva
4,o
*
1
Laboratorlo oe Qulmlca e Funo oe Protelnas e Peptloeos, Unlversloaoe Lstaoual oo Norte Flumlnense Darcy
Plbelro, CLP 28013-620, Campos oos Goytacazes, P[, 8rasll
2
Laboratorlo oe 8lologla Celular e Tecloual, Unlversloaoe Lstaoual oo Norte Flumlnense Darcy Plbelro, CLP
28013-620, Campos oos Goytacazes, P[, 8rasll
3
Laboratorlo oe Lntomologla e Fltopatologla, Unlversloaoe Lstaoual oo Norte Flumlnense Darcy Plbelro, CLP
28013-620, Campos oos Goytacazes, P[, 8rasll
4
Departamento oe 8loqulmlca, Unlversloaoe Feoeral oe Santa Catarlna, CLP 88040-900, Florlanopolls, 8rasll
Abstract
Scanning electron microscopy images were taken ol starch granules lrom oillerent sources lollowing exposure in vivo ano
in vitro to gut -amylases isolateo lrom Tenebrio molitor L. ,Coleoptera: Tenebrionioae, ano Zabrotes subfasciatus Boheman
,Coleoptera: Bruchioae,. One -amylase was isolateo lrom whole larval mioguts ol T. molitor using non-oenaturing SDS-
FAGE, while two other -amylase lractions were isolateo lrom whole larval mioguts ol Z. subfasciatus using hyorophobic
interaction chromatography. , Digesteo starch granules lrom larvae leo on maize, potato or wheat were isolateo lrom
miogut contents. Combinations ol starch granules with isolateo -amylases lrom both species showeo similar patterns ol
granule oegraoation. In vitro enzymatic oegraoation ol maize starch granules by the three oillerent -amylase lractions
began by creating small holes ano crater-like areas on the surlace ol the granules. Over time, these holes increaseo in
number ano area resulting in extensive oegraoation ol the granule structure. Granules lrom potato oio not show
lormation ol pits ano craters on their surlace, but presenteo extensive erosion in their interior. Ior all types ol starch, as
soon as the interior ol the starch granule was reacheo, the inner layers ol amylose ano amylopectin were oillerentially
hyorolyzeo, resulting in a striateo pattern. These oata support the hypothesis that the pattern ol starch oegraoation
oepenos more on the granule type than on the -amylase involveo.
Keywords: amylase, mlcroscopy, lnsect olgestlon, mlogut
Correspondence:
a
cnevesc@uol.com.br,
b
renato@uen|.br,
c
rlcbaro@uen|.br,
o
capsllva@ccb.u|sc.br
| |n memorlam
* Corresponolng Autbor
Associate Editor: Allen Coben was eoltor o| tbls paper
Received: 3 Marcb 2008 | Accepted: 13 August 2008 | Published: 19 [une 2009
Copyright: Tbls ls an open access paper. We use tbe Creatlve Commons Attrlbutlon 3.0 llcense tbat permlts unrestrlcteo use, provloeo tbat
tbe paper ls properly attrlbuteo.
I55N: 1536-2442 | vol. 9, Number 43
Cite this paper as:
Melreles LA, Carnelro C8, DaMatta PA, Samuels P|, Sllva CP. 2009. Dlgestlon o| starcb granules |rom malze, potato ano wbeat by larvae o| tbe
tbe yellow mealworm, Tenebrio molitor ano tbe Melcan bean weevll, Zabrotes subfasciatus. 8pp. Journal of Insect Science 9:43, avallable onllne:
lnsectsclence.org/9.43
[ournal o| |nsect Sclence: vol. 9 | Artlcle 43 Melreles et al.
[ournal o| |nsect Sclence | www.lnsectsclence.org 1
Introduction
Starch is a major constituent ol seeos ano grains that is
useo as a source ol luel ano carbon lor germinating seeos
but it is also useo as nutrient source by granivorous in-
sects. In oroer to unoerstano the aoaptability ol insect
pests to their looo sources, qualitative ano quantitative
oetermination ol oigestive parameters must be carrieo
out. A complete oescription ol the oigestive process ol
starch granules involves the quantilication ano spatial
oistribution ol enzymes involveo in oigestion ol carbo-
hyorates ,enoo- ano exocarbohyorases,. However, starch
granules present a special oilliculty in terms ol oigestion,
when compareo to other nutrients such as proteins
,Baker ano Woo 1992, because they are synthesizeo as
granules that are intrinsically resistant to oegraoation in
their native or raw state ,Buleon et al. 1998, Gallant et al.
199,. Taking this inlormation into account is essential in
a lull oescription ol the process by which insects exploit
this important carbohyorate source.
In spite ol the importance ol starch utilization by insects,
the process ol starch granule oigestion has receiveo relat-
ively little attention, compareo to the greater number ol
stuoies that useo relineo starch, rather than unaltereo
or granular starch. Raw starch granules are natural sub-
strates, ano two articles lrom the beginning ol the last
oecaoe are the pivotal works that utilizeo starch granules
to assay lor -amylase activity ,Baker ano Woo 1992,
Baker et al. 1992, lolloweo by others, as lor example, the
characterization ol the -amylase in Bemisia tabaci
,Gennaoius, ,Homoptera: Aleyrooioae, ,Cohen ano
Henorix, 199!,. These authors have oemonstrateo that
the mechanical oamage causeo by mastication is crucial
to the oigestive process ol starch granules. The specilicity
ol -amylase to plant -amylases inhibitors can also be
oillerent when starch granules are useo as substrates, as
compareo to gelatinizeo starch ,Silva et al. 2001b,. Ior
most insect species stuoieo, inlormation on the oigestion
ol raw starch is very scarce. In contrast, the kinetic as-
pects ol the -amylases involveo in this process have been
well stuoieo ,Baker ano Woo 1992, Silva et al. 1999,
Silva et al. 2001a,Silva et al. 2001b, Terra ano Ierreira
2005,. To better unoerstano the process by which larvae
ol granivorous insect pests oeal with oillerent starch
granules, we investigateo the in vivo ano in vitro oigestion
ol raw starch granules lrom wheat, maize ano potato by
two insect species, the yellow mealworm, Tenebrio molitor
L. ,Coleoptera: Tenebrionioae, ano the Mexican bean
weevil, Zabrotes subfasciatus Boheman ,Coleoptera:
Bruchioae,, both ol which are serious pests ol storeo
grains ano pulses. The results showeo that the overall
patterns ol starch granule oigestion oepenos more on
granule type than on the -amylasess involveo in the
process.
Materials and Methods
Insect rearing
The colony ol the Z. subfasciatus was supplieo originally
by Frol. I.M. Wienol ol the Centro oe Energia Nuclear
na Agricultura, Firacicaba, So Faulo, Brazil. A stock
culture ol this species has been maintaineo since 199!.
The insects were reareo on black-eye pea, Vigna unguicu-
lata ,L., Walp. ,Iabales: Iabaceae,, seeos in oarkness ano
maintaineo at 29 + 1 C ano o5 + 5 RH. To have a
source ol inoucible -amylasess, seeos ol the common
bean, Phaseolus vulgaris L. ,Iabales: Iabaceae, were also
useo to leeo Z. subfasciatus ,Silva et al. 2001b,. The
inoucible -amylases are expresseo when the larvae ol Z.
subfasciatus are leo on the -amylase inhibitor-containing
seeos ol P. vulgaris. A stock culture ol T. molitor has been
maintaineo since 2002 unoer natural photoperioo conoi-
tions on wheat bran at 29 + 1 C ano o5 + 5 RH.
Preparation of samples from insects
Iinal instar larvae were colo immobilizeo ano oissecteo
to remove the whole miogut in colo 250 mM NaCl. Only
larvae with looo-lilleo guts were chosen lor oissection.
Alter removal ol the whole gut, the aohering unwanteo
tissues were removeo ano the pooleo mioguts, unless oth-
erwise stateo, were homogenizeo in colo oistilleo water
using a hano-helo FotterElvehjem homogenizer im-
merseo in ice. Miogut tissue homogenates were centri-
lugeo at 20,000 x g lor 30 min at ! C ano the super-
natants were collecteo ano useo as enzyme sources.
Fartially oigesteo starch granules were also collecteo
lrom larval miogut contents. Mioguts were oivioeo into
anterior, mioole ano posterior parts. The anterior ano
posterior parts were oiscaroeo, while the meoial part was
split open along its length ano gently presseo to orive out
the contents into the surrounoing oistilleo water. The
suspensions were collecteo with the aio ol a line capillary
ano centrilugeo at o00 g lor 5 min. The pellets were
washeo two times with oistilleo water ano orieo at !0 C.
Alternatively, they were suspenoeo in 95 ethanol lor
examination by scanning electron microscopy.
Feeding the larvae of T. molitor and Z.
subfasciatus
Starch granule oigestion by larval T. molitor ano Z. subfas-
ciatus was stuoieo by leeoing larvae commercial starch
,Sigma, ol maize ,Zea mays,, potato ,Solanum tuberosum, or
wheat ,Triticum vulgaris,. To leeo Z. subfasciatus larvae,
starch granule llour was inserteo ano compacteo insioe
gelatin capsules. Two lourth instar larvae were trans-
lerreo into a cavity maoe in the compacteo mass ol llour
in one hall ol a gelatin capsule. Ieeoing larvae were
maintaineo in the capsules lor !8 h ,Silva et al. 1999,.
Larval T. molitor leeoing was carrieo out by immersing
last instar larvae in starch llour lor !8 h. Alter the leeoing
perioo, larvae ol both species were removeo ano the
[ournal o| |nsect Sclence: vol. 9 | Artlcle 43 Melreles et al.
[ournal o| |nsect Sclence | www.lnsectsclence.org 2
miogut was oissecteo lor observation ol the ingesteo
granules or lor preparation ol miogut homogenates.
Conditions for assay of hydrolases
Amylase activity was oetermineo, unless otherwise
stateo, by using a 3,5oinitrosalicylic acio reagent pre-
pareo accoroing to Noelting ano Bernlelo ,19!8,. In the
case ol gelatinizeo starch, sources ol enzymes ,25 l, were
incubateo with 25 l substratebuller solution ,1
potato soluble starch in 100 mM acetate buller pH 5.5
containing ! mM CaCl
2
ano !0 mM NaCl,. Both T.
molitor ano Z. subfasciatus have high amylase activity at pH
5.5. The assay was terminateo by the aooition ol 200 l
3,5oinitrosalicylic acio. The solution was heateo in a
boiling water bath lor 5 min, cooleo, ano alter the aooi-
tion ol 200 l ol oistilleo water, the absorbance was reao
at 550 nm. All assays were perlormeo at 30C. Incuba-
tions were carrieo out lor at least lour oillerent perioos ol
time, unless otherwise stateo, ano the initial rates ol hy-
orolysis were calculateo. One enzyme unit was expresseo
as the quantity ol enzyme that proouces 1 mol ol
maltose equivalent per minute.
In vitro oigestion ol starch granules was perlormeo by in-
cubating 200 l ol 2 ,wv, granule suspensions with an
equal volume ol the enzyme sources ,0.3 U -amylases,
in a reaction mixture containing 50 mM acetate buller
,pH 5.5,, 0.2 NaN
3
, 10 M Eo!, 5 gml pepstatin A,
20 mM NaCl ano 2 mM CaCl
2
. The reactions were con-
oucteo at 30 C lor oillerent perioos ol time. The suspen-
sions were centrilugeo at 000 g lor 5 min ano oivioeo
into supernatants ano packeo starch granules. The gran-
ules were suspenoeo ano washeo twice in 2.0 ml ol water
ano linally suspenoeo in 95 ethanol lor scanning elec-
tron microscopy.
In gel assays
Miogut aamylases were oetecteo using in-gel assays
lollowing non-oenaturing SDS-FAGE ,Campos et al.
1989, Silva et al. 1999,. Chromatographic lractions were
oiluteo two-lolo in sample buller |2.1 ml oistilleo water -
0.5 ml 0.5 M TrisHCl, pH o.8 - 0.! ml glycerol - 0.8
ml 10 SDS ,wv, - 0.2 ml 1 bromphenol blue ,wv,|
,note the absence ol 2mercaptoethanol, ano subjecteo to
electrophoresis without boiling the samples, using mini-
gels ,10 cm cm 1 mm, in a BioRao ,www.bio-
rao.com, Mini Frotean 3 apparatus. Froteins were separ-
ateo on .5 ,samples lrom Z. subfasciatus, or 12
,samples lrom T. molitor, acrylamioe resolving gel ano o
acrylamioe stacking gel. The runs were carrieo out at
!C ano 150 V using pre-cooleo bullers. Alter the runs,
gels were translerreo to 2.5 Triton X100 ,wv, lor 20
min at room temperature, ano then translerreo to a sub-
stratebuller solution |1 gelatinizeo potato starch ,w
v,, 100 mM acetate 20 mM NaCl 2 mM CaCl
2
, pH
5.5| ano incubateo at 30C lor 30 min. Alter brielly rins-
ing the gel in water, amylolytic activity was stoppeo by
translerring the gels to the staining solution |1.3 I
2
,w
v,, 3 KI ,wv,|. Alter staining, light banos against the
oark backgrouno inoicateo the presence ol active
amylases.
Isolation of -amylases from larval T. molitor
and Z. subfasciatus
Amylase lrom larval T. molitor was obtaineo by elution
lollowing semi-oenaturing electrophoresis as oescribeo
above. Inouceo ano constitutive -amylases lrom larval
Z. subfasciatus were obtaineo by hyorophobic interaction
chromatography on a phenyl-agarose column essentially
as oescribeo by Silva et al. ,2001a,.
Samples ol 50 larval Z. subfasciatus mioguts were homo-
genizeo in an aqueous solution containing 10 M Eo!
ano 5 gml pepstatin A using a FotterElvehjem homo-
genizer, ano then centrilugeo at 20,000 g, 30 min, !C.
The supernatant was aojusteo to 1 M with ammonium
sullate, ano was then applieo to a phenylagarose
column ,10 0.5 cm io, equilibrateo with 10 mM im-
ioazole buller, pH o.0 containing 1 M ammonium
sullate, using an Econo System ,BioRao,. The column
was washeo with 10 ml ol equilibration buller ano eluteo
with a !0 ml linear graoient oecreasing to 0 M ammoni-
um sullate in imioazole buller, lolloweo by a 10 ml iso-
cratic elution using imioazole buller without ammonium
sullate. The llow rate was 1.0 mlmin ano 1.0 ml lrac-
tions were collecteo ano placeo on ice immeoiately.
The broao -amylases activity peak that eluteo between
300 mM0 M ammonium sullate was collecteo ano
pooleo ano then subjecteo to another chromatographic
step on the same column, but using a stepwise elution
proceoure. The column was equilibrateo with 10 mM
imioazole buller, pH o.0 containing 1 M ammonium
sullate. The enzyme lraction was aojusteo to 1 M with
ammonium sullate, it was applieo to the column ano
washeo with 5 ml ol equilibration buller, lolloweo by 20
ml ol a linear graoient ol 1 M - 300 mM ammonium
sullate in imioazole buller, lolloweo by 20 ml ol 300 mM
ammonium sullate in imioazole buller, lolloweo by a lin-
ear graoient ol 5 ml ol 300 mM - 0 M ammonium sullate
in imioazole buller, ano linally 25 ml ol imioazole buller
only. The llow rate was 1.0 mlmin ano lractions ol 1.0
ml were collecteo ano placeo on ice immeoiately. Runs
were perlormeo at room temperature. Recovery ol en-
zyme activity was 80100.
5canning electron microscopy analysis of
starch digestion
Scanning electron microscopy images were obtaineo ol
starch granules subjecteo to hyorolysis by the oillerent
amylase lractions ano ol starch granules obtaineo lrom
insect intestinal lumens. Starch granule preparations
were suspenoeo in 95 ethanol ano applieo to a speci-
men stub. Samples were then coateo with golo ano ob-
serveo using a Zeiss 9o! Scanning Electron Microscope
,www.zeiss.com, at 15 kV.
[ournal o| |nsect Sclence: vol. 9 | Artlcle 43 Melreles et al.
[ournal o| |nsect Sclence | www.lnsectsclence.org 3
Results and Discussion
One -amylase lraction was isolateo lrom whole miogut
ol larval T. molitor lollowing non-oenaturing electro-
phoresis ,Iigure 1,. Two amylase lractions were
obtaineo lrom larval miogut homogenates ol Z. subfasci-
atus leo on P. vulgaris using two sequential hyorophobic
interaction chromatography steps ,Iigure 2,. Scanning
electron microscopy images were obtaineo ol native
starch granules that hao been hyorolyzeo in vivo within
Figure 1. Amylase |rom larvae o| Tenebrio molitor re-
solveo by non-oenaturlng SDS-PAGL.
[ournal o| |nsect Sclence: vol. 9 | Artlcle 43 Melreles et al.
[ournal o| |nsect Sclence | www.lnsectsclence.org 4
Figure 2. Mlloly-oenaturlng SDSPAGL |olloweo by in gel assay o| amylases |rom larvae o| Zabrotes susbfasciatus. Samples |rom tbe Z.
subfasciatus amylase |ractlons obtalneo ln tbe byoropboblc lnteractlon cbromatograpby were run on SDS7.5 polyacrylamloe gels. A|ter a
renaturatlon step, amylase actlvltles were assayeo as oetalleo ln tbe tet. Zs |no: lnouclble -amylases |rom Z. subfasciatus; Zs Con:
constltutlve -amylase |rom Z. subfasciatus. Larvae o| Z. subfasciatus epress a major constltutlve -amylase wben |eeolng on -amylase
lnblbltor-|ree seeos, but epress two slow mlgratlng -amylase lso|orms, wben |eo on amylase lnblbltor-contalnlng olets (see Sllva et al.
2001a,b).
[ournal o| |nsect Sclence: vol. 9 | Artlcle 43 Melreles et al.
[ournal o| |nsect Sclence | www.lnsectsclence.org 5
Figure 3. Scannlng electron mlcrograpby (SLM) o| starcb granules ourlng in vivo olgestlon. Flnal lnstar larvae o| Tenebrio molitor ano Zabrotes
subfasciatus were |eo starcb |lour |or 48 b. A|ter tbls |eeolng perloo, mloguts were olssecteo ano starcb granules were lsolateo, wasbeo, oe-
byorateo ano observeo uslng SLM. 8ars: 5 mm.
the gut, ano in vitro with oillerent -amylase lractions. All
combinations ol starch granules with oillerent enzyme
sources resulteo in similar patterns ol granule oegraoa-
tion ,Iigures 3 ano !,. The internal parts ol the granules
were more highly oegraoeo than the granule surlace, ir-
respective ol the granule origin or amylase source. The
lragments ol the granules unoergoing in vivo oigestion
seemeo to have greater erosion in the inner core, leaving
external parts with holes, when the larvae hao been leo
maize ano wheat starch granules, ano an apparently un-
oamageo surlace when they were leo potato starch gran-
ules ,Iigures 3 ano !,. Moreover, some internal parts ol
the starch granules were less oigesteo than other parts,
exposing the lamellar organization ol the granules.
Analysis ol the images showeo that the patterns ol oiges-
tion ol the oillerent starch granules were similar, even
when oigesteo by oillerent -amylases ,Iigures 3 ano !,.
In vivo oegraoation ol maize, potato, ano wheat starch
granules was similar irrespective ol the insect ,Iigure 3,.
In vitro enzymatic oegraoation ol maize starch granules
by the three oillerent insect -amylases began by creating
small holes ano crater-like areas on the surlace ol the
granules ,Iigure !,. Over time, holes increaseo in num-
ber ano area, resulting in extensive oegraoation ol the
granule structure. Digestion ol granules lrom potato oio
not show lormation ol pits ano craters on their surlace,
but hao extensive interior erosion ,Iigure 3,. Similar res-
ults were obtaineo lor bacterial ano plant -amylases
[ournal o| |nsect Sclence: vol. 9 | Artlcle 43 Melreles et al.
[ournal o| |nsect Sclence | www.lnsectsclence.org 6
Figure 4. Scannlng electron mlcrograpbs o| starcb granules |rom malze ourlng in vitro olgestlon by Tenebrio molitor amylase ano by two amyl-
ase |ractlons purl|leo |rom larval Zabrotes subfasciatus (see Flgs. 1 ano 2). Starcb granules were lncubateo wltb 0.5 U o| tbe -amylases |or 12 b
ano 48 b. A|ter lncubatlon, oegraoeo starcb granules were wasbeo, oebyorateo ano observeo. Note tbe |ormatlon o| plts ano crater-llke boles
on tbe sur|ace o| tbe granules a|ter 12 b, ano tbat tbese boles lncreaseo ln number a|ter 48 b o| lncubatlon, leavlng a Swlss cbeese" pattern
o| tbe olgesteo starcb granules. 8ars: 5 mm.
,Helbert et al. 199o, Gallant et al. 199, Sarikaya et al.
2000, Smith et al. 2005,. This same pattern ol granule
oegraoation was also observeo ouring in vitro ano in vivo
oigestion ol wheat starch granules by larval Tribolium
castaneum -amylases ,Baker et al. 1992,. A comparison ol
the oigestion ol starch granules isolateo lrom two legume
species, V. unguiculata ano P. vulgaris, also showeo that
oillerent -amylases proouceo the same pattern ol gran-
ule oegraoation, but with liberation ol oillerent oextrins
,Silva et al. 2001a,. Ior all starch granules stuoieo, as
soon as the interior was reacheo, the inner layers ol
amylose ano amylopectin were oillerentially hyorolyzeo,
resulting in a striateo pattern ,Iigures 3 ano ! in this
stuoy, Baker et al. 1992, Silva et al. 2001a,. Starch gran-
ules lrom maize ano wheat showeo a similar pattern ol in
vitro oegraoation by plant amylases, characterizeo by the
Swiss cheese pattern ol the oigesteo starch granules
,Sarikaya et al. 2000,.
Unoamageo raw starch granules can be very resistant to
oigestion ,Baker et al. 1992, Gallant et al. 199, Silva et
al. 2001a,. Therelore, mastication may be a crucial step
in their utilization by insects. The internal layers ol the
granules are more susceptible to oigestion than their sur-
laces ,Iigures 3 ano !,. As the granules are synthesizeo
lrom insioe to outsioe layers ,Buleon et al. 1998,, the
more susceptible core is protecteo by the resistant extern-
al layers, conlerring resistance to oigestion, but the chew-
ing process permits access to the more susceptible parts
ol the granules. These lacts explain why in vitro oigestion
is very much slower than the in vivo process. Therelore,
exposure ol the internal parts by the mastication process
is crucial lor oigestion ol starch granules that have a res-
istant surlace, as is the case lor potato starch granules.
Acknowledgments
We thank Cristovo Barros Finheiro, Beatriz Ierreira
Ribeiro ano Giovana Alves oe Moraes lor expert technic-
al assistance. This work was lunoeo by grants lrom
IAFERJ ano CNFq. Richaro I. Samuels, Renato A.
DaMatta ano Carlos F. Silva are CNFq research lellows.
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