Proceedinqs of the 7th Internatwnal Worhng Conference on Stored-product Protection - Volurne 2

The comparison of five determination methods for hiddeninsect infestation

Hau Xmgwei , Tan Xianchang and W u Jianchun'

Abstract

Five determmation methods for ludden msect infestation mcereals and pulses, reanng method, nmhydrin method, X-ray method, carbon dioxide method, and whole gramflotation method, were evaluated. Tests included four insectspecies, Sitoplulu« zearnais , Rhizopertha domuuca ,Sttotroga cerealella and Callosobruchus Chnnensis , threekmds of grain, wheat, nee, and mung bean. Threedifferent population densities of S zearnais were testedtoo The results showed that: based on the coefficient ofvariation and the relative error of maximum range, the bestmethod was reanng method (4.16,10.87 respectively), thenext was the carbon dioxide method (12.12, 29.72), andthe flotation method (38.72, 92.33) was the last one.Nmhydrin method (16.64, 42.19) and X-ray method(29.84, 70.24) had the smular efficacy. However, basedon the accuracy m number, the order from high to low IS:rearmg method> nmhydnn method> Xsray method >flotation method.

Introduction

There are some species of stored gram msects that can hvem the grain kernel, at least at their unmatured stages.These hidden msects hve and damage inside the grainkernels, make them difficult to be detected With the nakedeyes. After a penod of time, they develop and multiply,greatly increase their population, and cause senous damageto the gram especially when the gram IS stored for a longpenod. This problem has being concerned for a long time.Several determmation methods for hidden msect mfestationhave been developed (FAO, 1986, 1987; MIlner, 1950)The purpose of this paper IS to compare the efficacy of fivedifferent determmation methods for hidden msect infestationm gram: reanng method, nmhydrm method, X-ray method,carbon dioxide method, and flotation method

1Chengdu Gram Storage Research Institute, Mirnstry of InternalTrade, No 95 Huapaifang Street, Chengdu

Materials and Methods

Test insects and grains

Four hidden msect species, Siiophilus zeanuus( Motschusky ) , Rhizopertha dominuxi (FabricIUs) ,Siiotrcqa cerealella (OhvIer) and Cauoeooruch.u«chinensis (Lmnaeus) , three kmds of gram, wheat, nee,and mung bean were used, m addrtion, three populationdensities of S. zeamuus were tested for companng theeffects of population densities on the accuracy of different

methods

Preparation of infested grain

To obtain Immature stages, adults were held on 7 kg ofgrain for 20 - 25 days at 30, 65 - 70 % rho For S.cerealella and C. ch.mensis , smce they lay eggs for only afew days, the adults were added mto the grain about every 5- 7 days to insure a continual immature stages m the gram.

Test methods

Rearing method250 g gram sample was taken WIth a sample divider from

the msect infested gram, put the gram into a Jar, sealed Itwith a white cloth, placed the Jar into a controlledtemperature and humidity room (30, 65 - 70% rh ). Theadults were removed every 3 days begmmng at 42 days forS. zeomuus and S. cerealella, 49 days for R. dommica ,and 35 days for Colloeobruchu» chnmeneie .Carbon dioxide rnethodAll external msects were removed from the gram by

sieving after being held at 25, 70% rh for 24 hr. Gram wasplused mto a 500 ml Jar, shaken, and then the Jar wascompleted filled. The jar was sealed WIth a rubber septumand the net weight noted. The gram was held at 25 for 24hr , before a 10 ml gas sample was taken and analyzed usmga method smular to ISO 6639~4.X-ray methodAn X-ray apparatus (SOFTEX-TYPE S-E, Japan) with 5

x7 X-ray films (Tianjm sensitive matenal factory, Chma)was used. Infested grain was weighed 40g for wheat, 25gfor nee, 50g for mung bean, spread mto a layer of one gramthick on a hght-tight envelope containing film, and exposedunder the X-ray machine. The number of insects per kg was

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Proceedmqs of the 7 th Iniemational Working Conference on Stored-product Protection - Volume 2

calculated by counting the number of insects seen on thefilm.Nmhydrin methodThe detector was made by Chengdu Grain Storage

Research Institute. It consisted of two wheels that crushedthe grain into the nmhydrin treated paper. FIlter paper of3.3 mm width was soaked m 1% ninhydrin alcohol solutionand dried. 40 g for wheat, 25 g for rice, 60 g for mung beanwere passed through the detector slowly, and allowed toreact with nmhydrin in the paper for 30 min to 60 mm. Thepurple spots on the paper (one spot means one insect) werecounted, the number per kilogram grain calculated.Flotation methodFIve hundred grams were placed m an ammonium nitrate

solution with the density of 1.15 glml for wheat and bean,1.27 glml for nee. The solution was mixed at 1min

intervals to release air bubbles on the gram. MterlOminutes the floating grains were removed and desiccatedunder microscope to check for insects

Data analysis

All the tests were repeated 6 times. The efficacy ofmethods IS compared by standard error, coefficient ofvariation, range, relative error of maximum range. Relativeerror of maximum range was calculated by formula:Relative error of the maximum range = Range I insect

averate number

Results

All the results are listed m Table 1 - 8.

Table 1. Results of five determination methods at high population density of S. zeanuus in wheat.

Reanng X-ray Rearing X-ray Carbon dioxide

Average numberlkg 600 168.7 408.3 126 2.98

Standard error 28.3 44.6 30.3 37.6 6.6

Coefficient of variation (%) 4.7 26.4 7.4 29.8 20.05

Range 68 100 75 84 1.0

Relative error of maximum range% 11.33 59.28 18.37 66.67 33.52

Table 2. Results of five determmation methods at middle population density of S. zeamais m wheat.

Reanng X-ray Ninhydrin Flotation Carbon dIOXIde

Average number 131.3 95.8 75 44.43 2.67

Standard error 7.34 38.46 22.36 17.25 0.33

Coefficient of vanationt %) 5.59 40.13 29.81 38.72 12.53

Range 20 87.5 50 33.4 0.7

Relative error of maximum range% 15.23 91.31 66.67 75.0 26.25

Table 3. Results of five determination methods at low population density of S. zeamais in wheat.

Rearing X-ray Ninhydrin Flotation Carbon dioxide

Average number 87.33 45.83 100 49 0.938

Standard error 3.93 33.23 27.39 17.15 0.13

Coefficient of variationt %) 4.50 72.50 27.40 34.99 14.35

Range 12 75 75 42 0.36

Relative error of maximum range% 13.74 163.6 75 85.71 38.37

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Proceedings of the 7 th buematuma: Worktng Conference on Stored-product Protection - Volume 2

Table 4. Results of fivedeternunation methods at high population densityof S. zeamai» in rice.

Reanng X-ray Ninhydnn Flotation Carbon dioxide

Average number 1338 833.3 870 318.5 13.62Standard error 36.63 132.46 75.63 111.45 1.03Coefficient of variation/ %) 2.74 15.90 8.69 34.99 7.55Range 100 360 140 273 3.1Relative error of maximum range% 7.47 43.2 16.09 85.71 22.77

Table 5. Results of five determination methods at high population density of R. dommica m wheat.

Rearing X-ray Ninhydrin Flotation Carbon dioxide

Average number 666.7 337.5 475 105 2.62Standard error 27.21 70.26 122.47 82.78 0.29Coefficient of vanationf %) 4.08 20.82 25.78 78.84 10.92Range 72 200 325 209 0.8Relative error of maximum range% 10.8 59.26 68.42 199.05 30.53

Table 6. Results of fivedetermmation methods at high population density of S. cereolella in wheat

Rearing X-ray Ninhydnn Flotation Carbon dioxide

Average number 626.7 272.2 434.3 172.7 11.7Standard error 19.15 38.98 45.25 74.08 0.36Coefficient of variation/ % ) 3.06 14.32 10.42 42.90 3.06Range 45 67 126 210 1.0Relative error of maximum range% 7.18 24.62 32.28 121.62 8.55

Table 7. Results of five determination methods at high population density of C. chineneis m mung bean

Rearing X-ray Ninhydrin Flotation Carbon dioxide

Average number 1429.3 873.3 1260.8 494.5 11.93Standard error 64.33 164.28 87.22 61.42 1.96Coefficient of variation/ % ) 4.50 18.81 6.92 12.42 16.41Range 148 440 232 134 5.73Relative error of maximum range% 10.35 50.38 18.48 26.96 48.04

Reanng

Table 8. Total average results of fIve determmatIon methods at all insect speCIes, grain speCIes and population densities.

Ninhydrin X-ray Flotation Carbon dioxide

Coefficient of variation/ % )

Relative error of maximum range%

4.1610.87

16.6442.19

29.8470.24

38.9594.39

The stability of rearing method is the best among alltested methods. Its coefficient of vanation (%) for all thegrains, insect species is less than 5 % , and its relative errorof maximum range (%) IS less than 15 %, except those formiddle population densities in wheat, which are slightlyhigher than 5 % and 15 % ( 5.99 %, 15.23 %respectively) .

12.1229.72

From the results hsted in table 2 - 8, we can find thatcoefficient of variation and relative error of maximum range( %) of rearing method are the lowest of all five methods,ninhydrin method is lower than X-ray method, flotationmethod is the highest, and carbon dioxide is relative stable.In the view of average number we can find that: ninhydrinmethod is the closest one to reanng method, X-ray method

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Preceeduuje of the 7th Internatwnal Workvng Conference on Stored-product Protection - Yoiume 2

always lower than reanng method, however, nmhydnnmethod might be higher than reanng method especially atlow insect population density This might be because thererrught be false active reactions, or some of unmaturedinsects in the gram at the begmnmg of reanng do notsurvive to emerge. There IS a large difference between theaverage numbers m flotation method and the reanngmethod. According to the accuracy of number in this test,the order from high to low IS: reanng method > nmhydnnmethod> X-ray> flotation method. The carbon dioxide ISnot listed because It dose not directly reflect the number inthe grain (see discussion)

Discussion

The charactenstics of five methods are summanzed m Table9. Each method has ItS advantages and disadvantages.Reanng method might be the most accurate method, but isslow, smce It has to take at last 6 weeks to allow eachunmatured insect to develop and emerge from grams (ISO6639/3) . The other four methods are rapid methods andtake less than 2 days. The next accurate method is

mnhydnn method, It IS a good method for dry and small butnot fine grains. For large gram, It has to be partially brokenand might cause some msects to be lost, resulting in anunderestimation of the ludden infestation. In addition,ninhydrin method will not detect the dned dead insects, butWill give a positive reading for a dead insect whose bodyliquid IS not dried out. Gram with high moisture sometimeshows feint positive reaction, too. The effectiveness of X-ray method is SImilar to nmhydnn, but It needs someoperation skills and Its cost IS higher Flotation method wasthe least accurate, but It was cheaper and SImpler than othermothodsThe result of carbon dioxide method is expressed WIth the

carbon dioxide concentration m intergranular space, so, it isonly an mdirect measure of insect mfestation In addition,the carbon dioxide concentration m mtergranular space Itselfis affected by many factors, such as the shape of grain,vanety, the absorption of grain kernel to carbon dioxide,metabolism of gram, insect species and stage and mouldsTherefore, this estimation has to be rough, although Itsrepeatability IS better

Table 9. Characteristics of five deterrmnation methods for hidden insect infestation.

Effectiveness *

Method Speed Egg Large larva Small Pupa Comments Cost

Dead Alive Dead Ahve Larva Dead Ahve

Rearing slow D A D A A D A best moderate

Carbon dioxide rapid D C D A B D A good moderate

Ninhydrin rapid D B D A B D A good moderate

Flotation rapid D D B B C B B underestima te cheap

X-ray rapid C C A A B A A good expensive

* Notes: A-good, Bfair , C-bad, D-ml

Acknowledgment

We thank Dr Paul FIelds from Agnculture and Agn-FoodCanada, Wmnipeg Research Centre for reviewing andcorrecting the manuscnpt.

References

ISO 6639/1, 1986, Cereals and pulses- Determma tion ofhidden insect mfestation-Part 1: General pnnciples

ISO 6639/2, 1986, Cereals and pulses-Determmation ofhidden insect infestation-Part 2: Samplmg.ISO 6639/3, 1986, Cereals and pulses-Determmation ofhidden insect infestation-Part 3: Reference method.ISO 6639/4, 1987, Cereals and pulses-Determination ofhidden insect infestation-Part 4: Rapid methods.MIlne, M., Miolford , R L and Robert, K. 1950Application of X-ray techmque to the detection of internalinsect infestation of gram Journal of EconomicEntomology, 43, 933 - 935.

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