Fabres Et Al 2014

ORIGINAL PAPER

Comparative performance of the red flour beetle Triboliumcastaneum (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 flours. The

diets interfered differentially with development and insect

survival. Larvae size was reduced about 60 % in larvae fed

with V. unguiculata flour. 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

Tribolium castaneum (Coleoptera: Tenebrionidae), red flour

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 flowers, 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 Quımica e Funcao de Proteınas e Peptıdeos,

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: [email protected]

A. E. A. Oliveira

e-mail: [email protected]; [email protected]

123

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

influenced 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 identified, including phosphorus

(Chaudary and Lemonde 1962), thiamine, riboflavin, 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. castaneum fed with different diets composed of flours 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. castaneum is 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

Quımica e Funcao de Proteınas e Peptıdeos, Centro de

Biociencias e Biotecnologia, Universidade Estadual do

Norte Fluminense, Campos dos Goytacazes, RJ, Brazil.

The colony is maintained on a diet of wheat flour supple-

mented with whole meal flour and yeast extract (Himedia)

at 30 �C, 75 % R.H.

Seeds and flours

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 fine flour. Four wheat flours (Triticum aestivum L) were

commercially obtained in local markets from different

producers and were used in this work. Wheat flours were

kept at -70 �C for 12 h and subsequently maintained at

60� for 12 h. After cooling at room temperature the wheat

flours were sieved through a 300-lm mesh.

Larvae feeding and development

Adults were placed in wheat flour 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 flour. The diets

contained 100 % wheat flours 1, 2, 3 or 4 (W1, W2, W3 or

W4), P. vulgaris and V. unguiculata cotyledon flours (diets Pv

and Vu, respectively) and in some cases a mixture of 50 % of

P. vulgaris or V. unguiculata cotyledon flours and 50 % wheat

flour 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 flour were transferred to a new

container with the same wheat flour used during the larvae

stage. Pupae formed from larvae fed with Vu or Pv were

transferred to a new container with wheat flour 1. Emergence

adults were counted every 4 days during 58 DAH.

Feeding larvae with FITC-conjugated proteins

from legume flour

To unequivocally demonstrate the larval ingestion of diets

containing V. unguiculata and P. vulgaris flours, 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

123

centrifuged at 3409g for 5 min and supernatant proteins

were quantified (Bradford 1976). A fluorescein 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 flour (W2) at a 50 % ratio

(w/w) and used for feeding larvae. Control larvae were fed

with 100 % wheat flour (W2) without FITC. At 21 DAH,

larvae gut were dissected and analyzed by fluorescence

microscopy.

Larval triglycerides quantification

Triglycerides assay was performed using the ‘‘Triglicerides

120 kit’’ (Doles), following the manufacturer’s 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 quantification

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 quantification

Carbohydrates were quantified 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.

Quantification of protein, triglyceride

and carbohydrates in different diets

Proteins from diet flours were separated in albumin, glob-

ulin, prolamin and glutelin classes. To obtain albumin

fraction, flours 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 quantification of each fraction

was done by BCA method as described above (Smith et al.

1985).

Carbohydrates were quantified in supernatant obtained

after homogenization of flours 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 quantification by DNS method as described

above (Guglielminetti et al. 1999). Triglyceride was

quantified in supernatant obtained after homogenization of

different flours 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 quantification

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

123

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 significant 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 significant 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 flour had the highest concentra-

tion of albumin and wheat 2 flour had the highest glutelin

concentration (Table 1).

Changes in the insect diet also influenced significantly

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 0–24 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 flours

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 quantification in different cotyledons flours (ug/mg of flour)

Proteins (ug/mg of flour) Triglyceride (ug/mg of flour) Carbohydrate (ug/mg of flour)

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 significant difference between flours 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

123

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 flours. 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 flours, proteins from these

sources were extracted and conjugated with FITC. These

FITC-bound protein flours were mixed with wheat 2 (W2)

at a ratio of 50 % (w/w). Larvae midgut at 21 DAH were

dissected and visualized by fluorescence microscopy

(Fig. 4). Our results showed a high fluorescence in the

interior of larvae midgut proving the intake of V. unguic-

ulata (Fig. 4d) and P. vulgaris (Fig. 4f) flours 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 flours the levels of soluble protein, carbohydrates

and triglycerides were quantified in developing larvae

growing under diets containing 100 % of wheat 1 (W1),

wheat 2 (W2) or V. unguiculata (Vu) flours (Fig. 5). Since

no larva survived the treatment with 100 % P. vulgaris

flour 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 flour ? 50 % of

wheat 2 flour); Vu (100 % of Vigna unguiculata cotyledons flour);

Pv ? W2 (50 % of Phaseolus vulgaris cotyledons flour ? 50 % of

wheat 2 flour); Pv (100 % of Phaseolus vulgaris cotyledons flour).

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 significant

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

123

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 profile 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 flour 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 flour. 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 flour 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

profile 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 flour ? 50 % of wheat 2 flour); Vu (100 % of

Vigna unguiculata cotyledons flour); Pv ? W2 (50 % of Phaseolus

vulgaris cotyledons flour ? 50 % of wheat 2 flour); Pv (100 % of

Phaseolus vulgaris cotyledons flour). 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 significant 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)

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123

activity of 15 DAH larvae (a median value of activity) fed

with V. unguiculata flour showed a decrease in activity

compared with larvae fed with diet W2, but no significant

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 significant 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 flours. 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

flour 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

flour ? 50 % of wheat 2 flour). 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 flour ? 50 % of wheat 2 flour); Vu (100 % of Vigna

unguiculata cotyledons flour); Pv ? W2 (50 % of Phaseolus vulgaris

cotyledons flour ? 50 % of wheat 2 flour). Two-hundred eggs were

placed in flours, pupae and adults were counted over time. Results are

expressed as mean (±SEM) of three independent experiments.

Different letters indicate a significant 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

123

including T. castaneum, by reducing their offspring. Pro-

tein-rich fractions were seen to be more toxic than fibre and

starch fractions (Fields 2006). A mix of neem and P. sat-

ivum protein-rich flour 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 flour. 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

flour 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 fluorescence

microscopy (b, d and f). a–b Midgut of larva fed with W2 (920).

c–d Midgut of larva fed with 50 % of Vigna unguiculata FITC-

conjugated proteins ? 50 % of W2 flour (910). e–f Midgut of larva

fed with 50 % of Phaseolus vulgaris FITC-conjugated pro-

teins ? 50 % of W2 flour (910)

J Pest Sci

123

enormously (Renteria-Gutierez et al. 2000; Bostan and

Naeem 2002; Nehra et al. 1985; Ali et al. 2011). Our

results show a significant variation in developmental

parameters as larval growth, larvae survival, pupae for-

mation and adult emergency when the insect is fed on

different wheat flours. 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 significantly. 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 significantly

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 deficiency (Via 1999). T. castaneum males 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) quantifica-

tion in larvae fed with 100 % of wheat 1 (W1), wheat 2 (W2) or

Vigna unguiculata (Vu) flours 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 significant

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)

quantification

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

significant 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 significant differ-

ence and statistical treatments were performed using one-way

ANOVA test (df = 26, F = 63.56, P \ 0.0001)

J Pest Sci

123

(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, effi-

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 flour (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

flours we observed a high fluorescence 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 significant 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

significant 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

significant 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 significant

difference and statistical treatments were performed using one-way

ANOVA test (df = 26, F = 60.35, P \ 0.0001)

J Pest Sci

123

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 flour. These data show that

even small changes in diet composition have a reflection in the

profile 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 flour 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 form of

proteolytic digestion that was influenced by different soybean

cultivars (Naseri et al. 2010). Similar results were observed

when H. armigera larvae were fed with fresh legume pods,

vegetables, flowers 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 influenced 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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