ORI GI NAL PAPER

Comparative performance of the red our beetle Tribolium

castaneum (Coleoptera: Tenebrionidae) on different plant diets
Arianne Fabres

Janaina de Campos Macedo da Silva

Katia V. S. Fernandes

Jose Xavier-Filho

Gustavo Lazzaro Rezende

Antonia Elenir Amancio Oliveira
Received: 19 September 2013 / Accepted: 25 January 2014
Springer-Verlag Berlin Heidelberg 2014
Abstract Tribolium castaneum is a pest of stored foods
that causes serious economic losses. Understanding insect
nutritional requirements and the effects of different diets on
insect development can provide tools for developing
strategies to control insect and integrated pest management
systems. In this work we studied the performance of the T.
castaneum on different plant diets composed of Vigna
unguiculata, Phaseolus vulgaris and wheat ours. The
diets interfered differentially with development and insect
survival. Larvae size was reduced about 60 % in larvae fed
with V. unguiculata our. A delay in pupae formation was
also observed. The larval mortality of 100 % was observed
in the P. vulgaris diet. Carbohydrate and triglyceride
showed variations during development and with different
diets. Larvae fed with wheat diets showed a high concen-
tration of carbohydrates 21 days after hatching (DAH) and
triglyceride at 15 and 21 DAH. Larvae fed with wheat diets
showed a-amylase activity during development. At 15
DAH the activity of larvae fed with V. unguiculata
increased about 50 %. Cysteine protease activity in larvae
fed with wheat remained high throughout development.
Serine protease activity also varied according to diets.
These results show that the nutritional state of developing
larvae varies greatly with respect to triglycerides, carbo-
hydrate and digestive enzymes depending on the diet and
larval stage. Altogether, these results show that changes in
dietary nutrients can interfere with insect physiology.
Therefore changes in diet composition that may deprive
insects of essential nutrients or include toxic compounds
might be a good control strategy for stored product pests.
Keywords Tribolium castaneum Wheat
V. unguiculata P. vulgaris
Introduction
Triboliumcastaneum (Coleoptera: Tenebrionidae), red our
beetle, is a cosmopolitan pest of stored foods (Robinson
2005). This insect causes serious economic losses by
feeding on a wide diversity of foods such as broken grain,
milled grain products, cereals, meal, crackers, beans, spi-
ces, pasta, cake mix, dried pet food, dried owers, choco-
late and nuts (Via 1999). Adult females have a long life
span of approximately 3 years and can lay eggs in a rela-
tively continuous manner throughout their life (Howe
1962; Ziegler 1976). A previous work reported that T.
castaneum females oviposit about 1,000 eggs during their
lifetime (Rees 2004). T. castaneum infestation control is
done by phosphine fumigation (Jagadeesan et al. 2012);
however, acquisition of resistance to the substance has
been described for T. castaneum populations (Taylor and
Halliday 1986; Rajendran 2000; Benhalima et al. 2004;
Opit et al. 2012). Susceptibility of T. castaneum to insec-
ticide chlorfenapyr also varies enormously during the
insect lifecycle (Arthur and Fontenot 2012). Insecticide
resistance and human and environment toxicity problems
caused by the constant use of this chemical product have
Communicated by C. G. Athanassiou
A. Fabres J. de Campos Macedo da Silva
K. V. S. Fernandes J. Xavier-Filho G. L. Rezende (&)
A. E. A. Oliveira (&)
Laboratorio de Qu mica e Funcao de Prote nas e Pept deos,
Centro de Biociencias e Biotecnologia, Universidade Estadual
do Norte Fluminense Darcy Ribeiro-UENF, Av. Alberto Lamego
2000, P5, sala 224, Campos dos Goytacazes, RJ, Brazil
e-mail: grezende@uenf.br
A. E. A. Oliveira
e-mail: eleniramancio@yahoo.com.br; elenir@uenf.br
1 3
J Pest Sci
DOI 10.1007/s10340-014-0569-3
stimulated studies exploring alternative methods to insect
control (Nath et al. 2011). The study of the effects of dif-
ferent plant diets on insect life cycle and physiology can
contribute to developing of new control strategies such as
producing crops with endogenous resistance and integrated
pest management systems.
Plant-based diets may have different nutritional values and
these qualitative and quantitative differences on nutrients
composition may affect the rates of insect development
(Razmjou et al. 2013). Nutritional state is very important for
all T. castaneum development phases; however, the larvae
stage is particularly dependent on large quantities of food
(Robinson 2005). Previous work showed that T. castaneum
oviposition rate, growth and adult emergence are greatly
inuenced by food quality and composition (Ziegler 1976).
Previous work showed that a slight difference in food quality
considerably affected Tribolium sp. development (Campbell
and Runnion 2003). Food also affects the susceptibility of T.
castaneum and T. confusum to insecticidal pyrrolle chlorfe-
napyr. The presence of food increased insect resistance to
insecticide application (Arthur 2013). In addition, various
minerals and vitamins indispensable for the optimal growth of
T. confusum have been identied, including phosphorus
(Chaudary and Lemonde 1962), thiamine, riboavin, nico-
tinic acid, pyridoxine, pantothenic acid and biotin (Fraenkel
and Blewett 1947). Recent works have shown that the
expression of proteins and genes, especially those associated
with insect digestive processes, varies in response to changes
in food composition, showing that insect physiology is
affected by dietary compounds (Chi et al. 2009; Nogueira
et al. 2012).
In this work we studied the physiology and development of
T. castaneumfed with different diets composed of ours from
legume (Vigna unguiculata and Phaseolus vulgaris) cotyle-
dons and wheat. We particularly aimed at the analyses of
quantitative parameters relative to adults, larvae and pupae
growth and survival, as well as quantitative and qualitative
data on biochemical digestive measurements of insects sub-
jected to such diets. T. castaneumis an excellent insect model,
for which different molecular tools have been well-estab-
lished (Brown et al. 2009). Therefore, the study of the
development of this insect under different diets can originate
new tools for the control of insect infestation.
Materials and methods
Insects
Tribolium castaneum wild-type strain Georgia (GA-1) was
obtained from a colony maintained at the Laboratorio de
Qu mica e Funcao de Prote nas e Pept deos, Centro de
Biociencias e Biotecnologia, Universidade Estadual do
Norte Fluminense, Campos dos Goytacazes, RJ, Brazil.
The colony is maintained on a diet of wheat our supple-
mented with whole meal our and yeast extract (Himedia)
at 30 C, 75 % R.H.
Seeds and ours
Phaseolus vulgaris (red cultivar) and V. unguiculata
(fradinho cultivar) seeds were commercially obtained from
local markets (Campos dos Goytacazes, RJ, Brazil). Seeds
were maintained at -20 C for 3 days. After this period
seed coats were separated and the cotyledons were ground
to ne our. Four wheat ours (Triticum aestivum L) were
commercially obtained in local markets from different
producers and were used in this work. Wheat ours were
kept at -70 C for 12 h and subsequently maintained at
60 for 12 h. After cooling at room temperature the wheat
ours were sieved through a 300-lm mesh.
Larvae feeding and development
Adults were placed in wheat our 1(W1) during 24 h for
oviposition. Eggs obtained after 24 h were transferred to
different diets at the rate of 10 eggs/g of our. The diets
contained 100 % wheat ours 1, 2, 3 or 4 (W1, W2, W3 or
W4), P. vulgaris and V. unguiculata cotyledon ours (diets Pv
and Vu, respectively) and in some cases a mixture of 50 % of
P. vulgaris or V. unguiculatacotyledon ours and 50 %wheat
our 2 (diets Pv ? W2 and Vu ? W2, respectively). Larvae
fed with different diets were counted and larval lengths
measured 11 and 17 days after hatching (DAH) using the
software ImageJ. The total numbers of larvae measures
(including the three experiments) were: at 11 DAH,
W1 = 178; W2 = 141; W3 = 156; W4 = 123;
Vu ? W2 = 100; Vu = 85; Pv ? W2 = 100; Pv = 25; at
17 DAH, W1 = 169; W2 = 125; W3 = 148; W4 = 100;
Vu ? W2 = 78; Vu = 81; Pv ? W2 = 85. Pupae formed
were counted every 4 days during 58 DAH. Pupae formed
from larvae fed with wheat our were transferred to a new
container with the same wheat our used during the larvae
stage. Pupae formed from larvae fed with Vu or Pv were
transferred to a new container with wheat our 1. Emergence
adults were counted every 4 days during 58 DAH.
Feeding larvae with FITC-conjugated proteins
from legume our
To unequivocally demonstrate the larval ingestion of diets
containing V. unguiculata and P. vulgaris ours, proteins
from these sources were extracted and conjugated with
FITC. Soluble proteins from seed cotyledons were extrac-
ted using 0.1 M sodium phosphate, 0.15 M sodium chlo-
ride pH 7.0 M for 1 h at 4 C. The suspension was
J Pest Sci
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centrifuged at 3409g for 5 min and supernatant proteins
were quantied (Bradford 1976). A uorescein isothiocy-
anate (FITC) solution (50 mg/ml dimethyl sulfoxide) was
added to supernatants (1 lg FITC/110 lg protein) and the
mixture was kept for 16 h at 4 C. Unbound FITC was
removed by dialysis (dialysis membrane used retains pro-
teins of relative molecular masses of 8,000 Da or greater)
against distilled water for 24 h at 4 C and the resulting
solution was freeze-dried. The cotyledonary proteins-FITC
complex was mixed with wheat our (W2) at a 50 % ratio
(w/w) and used for feeding larvae. Control larvae were fed
with 100 % wheat our (W2) without FITC. At 21 DAH,
larvae gut were dissected and analyzed by uorescence
microscopy.
Larval triglycerides quantication
Triglycerides assay was performed using the Triglicerides
120 kit (Doles), following the manufacturers recom-
mendations. Larvae at 9, 11, 13, 15, 17, 19 and 21 DAH
were homogenized in 0.05 % Tween at a ratio of 1:100 (w/
v), shaken for 1 h at room temperature and centrifuged at
2,0009g for 1 min (Hildebrandt et al. 2011). An aliquot of
15 ll of supernatant was used for the assay which was run
at 37 C for 10 min. Absorbance of resulting solutions
were read at 510 nm. Concentration was determined using
a standard curve of glycerol.
Larval protein quantication
Protein concentration was determined by bicinchoninic
acid (BCA) method (Smith et al. 1985). Larvae at 9, 11, 13,
15, 17, 19 and 21 DAH were homogenized in 50 mM
potassium phosphate buffer pH 6.8 at a ratio of 1:100 (w/
v), shaken for 1 h at 4 C and centrifuged at 2,0009g for
1 min. An aliquot of 5 ll of supernatant was added to the
BCA reagent (Sigma) and incubated at 37 C for 30 min.
Absorbance readings were taken at 560 nm. Concentration
was determined using a standard curve of bovine serum
albumin (BSA).
Larval carbohydrates quantication
Carbohydrates were quantied according Guglielminetti
et al. (1999). Larvae at 9, 11, 13, 15, 17, 19 and 21 DAH
were homogenized in 50 mM potassium phosphate buffer
pH 6.8 at a ratio of 1:100 (w/v), shaken for 1 h at 4 C and
centrifuged at 2,0009g for 1 min. An aliquot of 5 ll of
supernatant was added to 200 ll of dinitrosalicylic acid
(DNS) and the mixture was boiled for 5 min. Absorbance
readings were taken at 440 nm. Concentration was deter-
mined according to a maltose standard curve.
Quantication of protein, triglyceride
and carbohydrates in different diets
Proteins from diet ours were separated in albumin, glob-
ulin, prolamin and glutelin classes. To obtain albumin
fraction, ours were homogenized in water at a ratio of
1:10 (w/v), during 30 min at 4 C and centrifuged at
12,0009g for 15 min. The supernatant was designated as
albumin and the pellet was homogenized in 0.1 M sodium
phosphate, 0.5 M sodium chloride, pH 7.6 at a ratio of 1:10
(w/v) for 30 min at 4 C and centrifuged at 12,0009g for
15 min. The supernatant (globulin) was separated and the
pellet was diluted in 70 % ethanol at a ratio of 1:10 (w/v),
shaken for 30 min at 4 C and centrifuged at 12,0009g for
15 min to obtain prolamin fraction (supernatant). The
pellet was homogenized in 0.1 M NaOH at a ratio of 1:10
(w/v), shaken for 30 min at 4 C and centrifuged at
12,0009g for 15 min. The supernatant obtained was des-
ignated as glutelin. Protein quantication of each fraction
was done by BCA method as described above (Smith et al.
1985).
Carbohydrates were quantied in supernatant obtained
after homogenization of ours with 0.5 M potassium
phosphate buffer pH 6.8 at a ratio of 3:100 (w/v). The
mixture was shaken for 1 h at 4 C and centrifuged at
2,0009g for 1 min; 20 ll of the supernatant were used for
carbohydrate quantication by DNS method as described
above (Guglielminetti et al. 1999). Triglyceride was
quantied in supernatant obtained after homogenization of
different ours with 0.05 % Tween at a ratio of 3:100 (w/
v). The mixture was shaken for 1 h at room temperature
and centrifuged at 2,0009g for 1 min (Hildebrandt et al.
2011); 25 ll of the supernatant were used in quantication
by Triglicerides 120 kit (Doles) as described above.
Larval cysteine protease activity
Cysteine protease activity was determined according to
Michaud et al. (1994) using azocasein as substrate and a
curve of pure papain as standard. Larvae at 9, 11, 13, 15,
17, 19 and 21 DAH were homogenized in buffer (100 mM
sodium phosphate, 100 mM citrate phosphate, 0.1 %, triton
X-100 and 1.5 mM DTT, pH 5.6) at a ratio of 1:185 (w/v).
The larval extract was shaken for 1 h at 4 C and centri-
fuged at 4,0009g for 5 min. An aliquot of 30 ll of
supernatant was incubated with 80 ll of 1 % azocazein at
37 C for 1 h. An aliquot of 300 ll of TCA (10 %) was
J Pest Sci
1 3
added to stop the reaction. Solutions were pulse-speed
centrifuged and 300 ll of 1 M NaOH was added to the
supernantant. Absorbance readings were taken at 440 nm.
Larval serine protease activity
Serine protease activity was determined according to De-
martini et al. (2007) using BApNa as substrate and pure
trypsin activity as standard. Larvae at 9, 11, 13, 15, 17, 19
and 21 DAH were homogenized in 50 mM Tris-HCl buffer
pH 8.0 at a ratio of 1:110 (w/v), shaken for 1 h at 4 C and
centrifuged at 4,0009g for 5 min. An aliquot of 50 ll of
supernatant was incubated with 38 ll of 5 mM BApNa and
212 ll of 100 mM Tris-HCl buffer, pH 8.0 at 37 C for
1 h. The reaction was stopped with 150 ll of 30 % acetic
acid and absorbance readings were taken at 440 nm.
Larval a-amylase activity
a-Amylase activity was determined according to Gugliel-
minetti et al. (1999). Larvae at 9, 11, 13, 15, 17, 19 and 21
DAH were homogenized in 50 mM potassium phosphate
buffer pH 6.8 at a ratio of 1:200 (w/v), shaken for 1 h at
4 C and centrifuged at 4,0009g for 5 min. Larval
homogenate supernatant (5 ll) was incubated with 6 ll of
1 % starch and 95 ll of 50 mM sodium phosphate buffer
pH 6.8 at 37 C for 45 min. An aliquot of 200 ll of DNS
was added and the mixture was boiled for 5 min. Absor-
bance readings were taken at 440 nm and the activity was
determined according to a standard curve of maltose.
Statistical analysis
For all experiments, mean and standard deviation were
calculated. Statistical analysis of one-way analysis of var-
iance (ANOVA) followed by Tukey test (P\0.05) were
performed using GraphPad Prism 5.0.
Results
Composition of distinct diets and effects on T.
castaneum survival, larval and pupal development
Composition of the diets showed signicant differences in
levels of carbohydrates in all diets and V. unguiculata and
P. vulgaris diets showed the highest concentrations. The
levels of triglyceride were also higher in these legume
diets. A signicant variation in quality and quantity of
protein was observed (Table 1). V. unguiculata and P.
vulgaris cotyledons are rich in protein, mainly albumin and
globulins. Wheat cultivars showed lower concentrations of
proteins and between the four protein classes, glutelins
were the most concentrated. Among the four wheats used
in this work, the wheat 1 our had the highest concentra-
tion of albumin and wheat 2 our had the highest glutelin
concentration (Table 1).
Changes in the insect diet also inuenced signicantly
the larval survival at 11 and 17 DAH (Fig. 1). The highest
larval mortality was observed in larvae fed with W4 and Pv
diets (Fig. 1a, b). The larval mortality in the W4 diet at 17
DAH was about 56 % while in the Pv diet it was approx-
imately 86 % at 11 DAH and 100 % at 17 DAH (Fig. 1a,
b).
To analyze T. castaneum larval growth under different
plant diets, eggs of 024 h were placed in distinct diets.
Larvae sizes were measured at 11 and 17 DAH (Fig. 2). A
higher larval growth was observed in wheat 1 diet (W1)
while larvae fed on the other diets, including those based
on other wheats (W2, W3 and W4), showed size reduction
at 11 and 17 DAH. The diets containing 50 and 100 % of
V. unguiculata (Vu ? W2 and Vu, respectively) and P.
vulgaris (Pv ? W2 and Pv, respectively) cotyledon ours
were the most effective in reducing larval size. The larvae
fed with 50 % of V. unguiculata and P. vulgaris mixed
with wheat 2 (W2) showed size reduction at 11 DAH of 34
and 22 %, respectively, when compared to larvae fed with
Table 1 Albumins, globulins, prolamins, glutelins, triglycerides and carbohydrates quantication in different cotyledons ours (ug/mg of our)
Proteins (ug/mg of our) Triglyceride (ug/mg of our) Carbohydrate (ug/mg of our)
Albumin Globulin Prolamin Glutelin
W1 32.51 (0.13)
a
16.32 (0.84)
a
5.51 (0.26)
a
84.04 (1.68)
a
0.42 (0.025)
a
15.3 (0.30)
a
W2 15.96 (0.75)
a,c
13.45 (1.71)
a
7.67 (0.04)
a,c
85.65 (0.53)
a
0.32 (0.01)
a
14.47 (0.07)
b
W3 1.26 (0.66)
b
8.53 (0.73)
a
8.96 (0.63)
b,c
33.88 (0.68)
b
0.37 (0.014)
a
12.97 (0.05)
c
W4 12.08 (1.37)
c
10.2 (1.53)
a
14.64 (0.51)
d
40.04 (0.94)
b,c
0.46 (0.041)
a
13.32 (0.046)
c
Vu 627.7 (7.11)
d
89.92 (11.2)
b
4.50 (0.38)
a
44.77 (0.75)
c,d
0.96 (0.002)
b
15.46 (0.07)
d
Pv 493.5 (8.88)
e
97.65 (1.86)
b
5.00 (0.24)
a
47.8 (0.66)
d
0.95 (0.011)
b
16.07 (0.14)
d
Results are expressed as mean (SEM) of three independent experiments. Different letters indicate a signicant difference between ours in the
same molecule and statistical treatments were performed using one-way ANOVA test (df = 83, F = 100.3, P\0.0001)
J Pest Sci
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W2. Diets containing 100 % of V. unguiculata and P.
vulgaris decreased larval size 56 and 60 %, respectively,
when compared with the W1 diet (Fig. 2a). So, feeding of
Tribolium on exclusively legume-based diets was more
deleterious to larval development than when mixing these
with the naturally-preferred wheat ours. At 17 DAH lar-
vae fed with diets containing 100 % of V. unguiculata
showed decreased size of 60 %, when compared to W1 diet
(Fig. 2b). Images of representative larvae are shown in
Fig. 2c.
Pupae formed in different diets during the experiment
time were also evaluated (Fig. 3a, b) and the results
showed a delay in pupae formation when diet W1 was
compared with all other diets. At 18 DAH pupae formation
was observed (44 pupae) only in diet W1, indicating that
this wheat diet favored pupae formation. The longest
delays in pupae formation were observed in diets W4
(Fig. 3a) and Vu (Fig. 3b). Adults emergence was also
evaluated during 58 DAH (Fig. 3c, d) and the highest
reduction rates in adult emergence were observed for both
larvae and pupae developed in W2 and Vu diets (Fig. 3c,
d).
To demonstrate the larval ingestion of diets containing
V. unguiculata and P. vulgaris ours, proteins from these
sources were extracted and conjugated with FITC. These
FITC-bound protein ours were mixed with wheat 2 (W2)
at a ratio of 50 % (w/w). Larvae midgut at 21 DAH were
dissected and visualized by uorescence microscopy
(Fig. 4). Our results showed a high uorescence in the
interior of larvae midgut proving the intake of V. unguic-
ulata (Fig. 4d) and P. vulgaris (Fig. 4f) ours by the
larvae.
Effects of different diets on nutritional state
of T. castaneum larvae
To evaluate the nutritional status of larvae fed with dif-
ferent ours the levels of soluble protein, carbohydrates
and triglycerides were quantied in developing larvae
growing under diets containing 100 % of wheat 1 (W1),
wheat 2 (W2) or V. unguiculata (Vu) ours (Fig. 5). Since
no larva survived the treatment with 100 % P. vulgaris
our at 17 DAH the effect of this diet on larval nutritional
state was not analyzed. The higher concentration of pro-
teins was observed in larvae at 9 DAH for all analyzed
diets. After this time the levels of proteins decreased about
50 % at 11 DAH and remained relatively constant during
larval development. Total proteins do not change in larvae
fed with different diets (Fig. 5a).
Total carbohydrate showed considerable variations
during larval development as well as in larvae fed with
different diets (Fig. 5b). The higher concentrations during
larval development were observed at 21 DAH in larvae fed
with wheat 1 (W1) and wheat 2 (W2). The main observed
change in different diets was a low sugar concentration
detected in larvae fed with 100 % of V. unguiculata at 9,
11, 15 and 21 DAH (Fig. 5b).
The triglycerides concentration varied greatly during
larval development and with different diets (Fig. 5c).
Larvae fed with wheat 1 (W1) showed the highest con-
centration of triglyceride at 15 and 21 DAH. The lowest
triglyceride levels were observed in larvae fed with 100 %
V. unguiculata (Vu) during all larval development
(Fig. 5c). These results show that the nutritional state of
developing T. castaneum larvae varies greatly with respect
Fig. 1 Survival of larvae at 11 (a) and 17 DAH (b) fed with different
plant diets: W1, W2, W3 and W4 (100 % of wheat 1, 2, 3 or 4);
Vu ? W2 (50 % of Vigna unguiculata cotyledons our ? 50 % of
wheat 2 our); Vu (100 % of Vigna unguiculata cotyledons our);
Pv ? W2 (50 % of Phaseolus vulgaris cotyledons our ? 50 % of
wheat 2 our); Pv (100 % of Phaseolus vulgaris cotyledons our).
The calculation of the percentage was made in relation to the number
of initial eggs. Results are expressed as mean (SEM) of three
independent experiments. Different letters indicate a signicant
difference and statistical treatments were performed using one-way
ANOVA test for larvae 11 DAH (df = 23, F = 45.15, P\0.0001)
and for larvae 17 DAH (df = 23, F = 58.06, P\0.0001)
J Pest Sci
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to triglycerides and carbohydrate depending on the diet and
larval stage.
Effects of different diets on the activity of digestive
enzymes of the larvae
The activity of a-amylase was determined along larval
development in larvae fed with diets W1 and W2 (wheat
diets). T. castaneum larvae displayed a-amylase activity
during all larval development, with the activity being
higher between 9 and 13 DAH, and decreasing after this
period (Fig. 6a). The enzyme activity prole was similar in
larvae fed with both wheat diets (1 and 2), except at 19 and
21 DAH where the activity values were lower in larvae fed
with wheat 2 (Fig. 6a). The a-amylase activity of larvae at
15 DAH (a median value of activity for diets W1 and W2)
fed with 100 % V. unguiculata our increased about 50 %
when compared to that from larvae fed with W1 and W2
(Fig. 6b).
Cysteine protease activity was determined in larvae fed
with wheat diets (W1 and W2) and 100 % of V. unguicu-
lata our. In larvae fed with wheat diets the activity was
high since the 9 DAH and remained relatively high
throughout development. At 11, 13 and 17 DAH the
activity was higher in larvae fed with W2 (Fig. 7a).
Enzyme activity in larvae at 15 DAH (a median value of
activity) fed with 100 % of V. unguiculata our did not
differ statistically from activity of larvae fed with wheat
diets W1 and W2 (Fig. 7b).
Serine protease activity was also measured and the
prole was relatively similar in larvae fed with wheat diets
W1 and W2 until 13 DAH. At 15 and 17 DAH the activity
decreased in larvae fed with W1 (Fig. 8a). Serine protease
activity of larvae feeding on diet W2 increased at 15 and 17
DAH and then decreased until the 21 DAH. Serine protease
Fig. 2 Size analysis of larvae fed with different plant diets: W1, W2,
W3 and W4 (100 % of wheat 1, 2, 3 or 4); Vu ? W2 (50 % of Vigna
unguiculata cotyledons our ? 50 % of wheat 2 our); Vu (100 % of
Vigna unguiculata cotyledons our); Pv ? W2 (50 % of Phaseolus
vulgaris cotyledons our ? 50 % of wheat 2 our); Pv (100 % of
Phaseolus vulgaris cotyledons our). Larval size was measured
11 days after hatching (DAH) (a) and 17 DAH (b). c Representative
image of larvae 17 DAH. Scale bar = 1 mm. Results are expressed as
mean (SEM) of the total number of larvae measures (including the
three experiments). At 11 DAH (n W1 = 178; W2 = 141;
W3 = 156; W4 = 123; Vu ? W2 = 100; Vu = 85;
Pv ? W2 = 100; Pv = 25). At 17 DAH (n, W1 = 169;
W2 = 125; W3 = 148; W4 = 100; Vu ? W2 = 78; Vu = 81;
Pv ? W2 = 85). Different letters indicate a signicant difference
and statistical treatments were performed using one-way ANOVA test
for larvae 11 DAH (df = 907, F = 245.9, P\0.0001) and for larvae
17 DAH (df = 789, F = 271.1, P\0.0001)
J Pest Sci
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activity of 15 DAH larvae (a median value of activity) fed
with V. unguiculata our showed a decrease in activity
compared with larvae fed with diet W2, but no signicant
difference compared to those fed with diet W1 (Fig. 8b).
Discussion
Insects in general can develop on foods containing differ-
ent levels of nutrients; however, optimal growth requires a
balanced diet that varies among insect species. Variations
in the quantity or quality of an acceptable diet can have
profound effects on insect development (Chapman 1998).
In this work we observed a considerable variation in larval
development, pupae formation and insect survival in sev-
eral diets, including different wheats. The tested diets
showed signicant variations in some nutrient composition.
The V. unguiculata and P. vulgaris bean-based diets
showed the higher concentrations of carbohydrates, tri-
glycerides and proteins, mainly of albumin- and globulin-
type reserve proteins. Unlike legume beans, wheat diets
showed lower concentrations of total protein, including
albumin and globulin and the glutelin proteins were the
most concentrated in wheat ours. Previous literature
reported the toxicity of seed protein-rich fractions to insect
pests (Hou et al. 2004a, b; Fields 2006). Pisum sativum
protein-rich fraction mixed with wheat kernels or wheat
our showed to be toxic to nine storage grain beetles,
Fig. 3 Pupae and adults formed along time in larvae fed with
different plant diets. a Pupae formed from larvae fed with W1, W2,
W3 and W4 (100 % of wheat 1, 2, 3 or 4). b Pupae formed from
larvae fed with Vu ? W2 (50 % of Vigna unguiculata cotyledons
our ? 50 % of wheat 2 our). c Adults formed from larvae fed with
W1, W2, W3 and W4 (100 % of wheat 1, 2, 3 or 4). d Adults formed
from larvae fed with Vu ? W2 (50 % of Vigna unguiculata
cotyledons our ? 50 % of wheat 2 our); Vu (100 % of Vigna
unguiculata cotyledons our); Pv ? W2 (50 % of Phaseolus vulgaris
cotyledons our ? 50 % of wheat 2 our). Two-hundred eggs were
placed in ours, pupae and adults were counted over time. Results are
expressed as mean (SEM) of three independent experiments.
Different letters indicate a signicant difference between larvae with
same age and statistical treatments were performed using one-way
ANOVA test for pupae from larvae fed with W1, W2, W3 and W4
(df = 131, F = 11.8, P\0.0001), for pupae from larvae fed with
W2, Vu ? W2, Vu and Pv ? W2 (df = 131, F = 67.16,
P\0.0001), for adults from larvae fed with W1, W2, W3 and W4
(df = 131, F = 8.19, P\0.0001) and for adults from larvae fed with
W2, Vu ? W2, Vu and Pv ? W2 (df = 131, F = 43.86,
P\0.0001)
J Pest Sci
1 3
including T. castaneum, by reducing their offspring. Pro-
tein-rich fractions were seen to be more toxic than bre and
starch fractions (Fields 2006). A mix of neem and P. sat-
ivum protein-rich our acted synergistically against T.
castaneum (Hou et al. 2004a). The combinations of P.
sativum protein-rich fraction and parasitoids reduced the
populations of the rice weevil Sitophilus oryzae and the
rusty grain beetle Cryptolestes ferrugineus by 98 and 75 %,
respectively (Hou et al. 2004b).
Literature data have also shown that T. castaneum beetle
can develop in several diets, but some diets greatly favor
insect development, such as wheat our. Although wheat is
the T. castaneum preferred food, the level of infestation of
different wheat cultivars by T. castaneum varies
Fig. 4 Microscopy of larvae midgut at 21 DAH fed with W2 (wheat
our 2) or Vigna unguiculata and P. vulgaris FITC-conjugated
proteins mixed with W2. Larvae midgut at 21 DAH were dissected
and visualized by optical microscopy (a, c and e) and uorescence
microscopy (b, d and f). ab Midgut of larva fed with W2 (920).
cd Midgut of larva fed with 50 % of Vigna unguiculata FITC-
conjugated proteins ? 50 % of W2 our (910). ef Midgut of larva
fed with 50 % of Phaseolus vulgaris FITC-conjugated pro-
teins ? 50 % of W2 our (910)
J Pest Sci
1 3
enormously (Renteria-Gutierez et al. 2000; Bostan and
Naeem 2002; Nehra et al. 1985; Ali et al. 2011). Our
results show a signicant variation in developmental
parameters as larval growth, larvae survival, pupae for-
mation and adult emergency when the insect is fed on
different wheat ours. The larval mortality in the W4 diet
at 17 DAH was about 56 % and a delay in pupae formation
was also observed. Adult formation was also severely
impaired by the W2 diet. These data reveal that T. casta-
neum is extremely sensitive to changes in food
composition.
Ming and Cheng (2012) showed that T. castaneum
males fed with different amounts of nutrients had their
larval and pupal weight, developmental time and adult
body weight varying signicantly. The enrichment of T.
castaneum diets with yeast improved the insect develop-
ment (Sokoloff et al. 1966).
Wong and Lee (2011) showed a relationship between
the amount of protein and carbohydrates in the diets and
the adult emergency rate. Adult emergences signicantly
increased in diets rich in proteins and decreased in diets
with high carbohydrate content.
Larvae egg cannibalism also varied according to the diet
and the author suggested that this behavior may be a result of
some nutrient deciency (Via 1999). T. castaneummales fed
with a low nutritional composition diet reduced the endog-
enous production of the pheromone 4, 8-dimethyldecanal
Fig. 5 Protein (a), carbohydrates (b) and triglyceride (c) quantica-
tion in larvae fed with 100 % of wheat 1 (W1), wheat 2 (W2) or
Vigna unguiculata (Vu) ours 9, 11, 13, 15, 17, 19 and 21 days after
hatching (DAH). Results are expressed as mean (SEM) of three
independent experiments. Different letters indicate a signicant
difference between larvae with same age, and statistical treatments
were performed using one-way ANOVA test for protein (df = 125,
F = 102.9, P\0.0001), for carbohydrate (df = 125, F = 48.76,
P\0.0001) and for triglycerides (df = 83, F = 13.22, P\0.0001)
quantication
Fig. 6 a-Amylase activity of larvae during development fed with
different plant diets. a a-Amylase activity of larval extract at 9, 11,
13, 15, 17, 19 and 21 days after hatching (DAH) fed with wheat 1
(W1) or 2 (W2). b a-Amylase activity of larval extract at 15 DAH fed
with W1, W2 or Vigna unguiculata (Vu). Results are expressed as
mean (SEM) of three independent experiments. Asterisk indicates a
signicant difference between larvae of the same age, and statistical
treatments were performed using one-way ANOVA test (df = 137,
F = 9.86, P\0.0001). Different letters indicate a signicant differ-
ence and statistical treatments were performed using one-way
ANOVA test (df = 26, F = 63.56, P\0.0001)
J Pest Sci
1 3
(Ming and Lewis 2010). Dietary stress, reduction in dietary
quality or dietary deprivation caused lower weights, delayed
adult emergence and contributed to increase T. castaneum
fungus infestation (Lord 2010). Essential oils from Chry-
santhemum species leaves affected T. confusum nutritional
indices relative growth rate, relative consumption rate, ef-
ciency of conversion of ingested food and feeding deterrence
index (Haouas et al. 2012).
Life cycle parameters, such as extent of the larval and
pupal periods, pre-adult development time and female
fecundity of Helicoverpa armigera were also affected by
different diets, containing varieties of chickpea, beans or
tomato (Razmjou et al. 2013). Wheat our (Triticum aes-
tivum L.), date fruits (Phoenix dactylifera L.), sorghum
(Sorghum vulgare L.) and barley (Hordeum vulgare L.)
interfered in different ways in Plodia interpunctella larval
weight, mortality, pupation and adult emergence (Bouayad
et al. 2008).
When larvae were fed diets containing V. unguiculata
and P. vulgaris FITC-labeled proteins mixed with wheat
ours we observed a high uorescence in the larvae mid-
gut. These results show that T. castaneum does not dis-
criminate between dietary components coming from
different origins, feeding well on such mixed grain diet. So
the control strategy of mixing plant toxic compounds with
insect food can be more effective in controlling this pest
infestation than the topical application of insecticide.
Morris et al. (2009) established, through proteomic ana-
lysis, a gut ranking of midgut proteins of T. castaneum larvae
and showed that a-amylase is highly expressed in this tissue.
Previous work showed that T. castaneum protein digestion is
performed mainly by cysteine proteases in the acidic anterior
midgut, with minor contributions of serine proteases, in an
Fig. 7 Cysteine protease activity of larvae during development fed
with different plant diets. a Cysteine protease activity of larval extract
at 9, 11, 13, 15, 17, 19 and 21 days after hatching (DAH) fed with
wheat 1 (W1) or 2 (W2). b Cysteine protease activity of larval extract
at 15 DAH fed with W1, W2 or Vigna unguiculata (Vu). Results are
expressed as mean (SEM) of three independent experiments.
Asterisk indicates a signicant difference between larvae of same
age and statistical treatments were performed using one-way ANOVA
test (df = 124, F = 7.76, P\0.0001). Different letters indicate a
signicant difference and statistical treatments were performed using
one-way ANOVA test (df = 26, F = 2.66, P\0.0001)
Fig. 8 Serine protease activity of larvae during development fed with
different plant diets. a Serine protease activity of larval extract at 9,
11, 13, 15, 17, 19 and 21 days after hatching (DAH) fed with wheat 1
(W1) or 2 (W2). b Serine protease activity of larval extract at 15 DAH
fed with W1, W2 or Vigna unguiculata (Vu). Results are expressed as
mean (SEM) of three independent experiments. Asterisk indicate a
signicant difference between larvae with same age and statistical
treatments were performed using one-way ANOVA test (df = 128,
F = 14.92, P\0.0001). Different letters indicate a signicant
difference and statistical treatments were performed using one-way
ANOVA test (df = 26, F = 60.35, P\0.0001)
J Pest Sci
1 3
alkaline posterior midgut (Vinokurov et al. 2009). Based on
these studies that showed a-amylase, cysteine and serine
proteases as the mainly T. castaneum larval digestive
enzymes, the activity of these enzymes was determined along
development in larvae fed with wheat and in larvae 15 DAH
fed with V. unguiculata. Our results showed that enzymes
activities ranged during larval development and in different
diets, including in different wheat our. These data showthat
even small changes in diet compositionhave a reectioninthe
prole digestive of the T. castaneum larvae. Changes in the
activity of digestive enzymes of insects in response to dif-
ferent diets have also been shown in literature (Wool et al.
1986; Bouayad et al. 2008; Kotkar et al. 2009; Naseri et al.
2010). T. castaneum a-amylase activity was increased in
adults when corn our diet was supplemented with yeast, but
this increase was not dose dependent (Wool et al. 1986).
Larvae of H. armigera showed a complex and diverse formof
proteolytic digestion that was inuenced by different soybean
cultivars (Naseri et al. 2010). Similar results were observed
when H. armigera larvae were fed with fresh legume pods,
vegetables, owers and cereal. Larval amylase and proteinase
activities change according to the contents of protein and
carbohydrate in the diet, suggested that H. armigera regulates
the levels of digestive enzymes in response to diet composi-
tion (Kotkar et al. 2009). Plodia interpunctella a-amylase
activity was also inuenced by diets. In larvae fed with
Phoenix dactylifera fruits, a reduction in a-amylase activity
was observed. The authors suggested that this reduction was
due to the high levels of glucose present in these fruits
(Bouayad et al. 2008).
The results obtained in this work showed that T. casta-
neum physiology was affected by change in foods. This
interference could be related to qualitative and/or quantita-
tive differences on nutrient composition. Therefore changes
in diet composition that may deprive insects of essential
nutrients or include compounds which are toxic to them
might be a good control strategy for stored product pests.
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