Life Science Journal, 2012;9(x)http://www.lifesciencesite.com

Haemolymph amino acids alterations in pyridalyl treated desert locust,Schistocerca gregaria in relation to age

Zahia, K. Mostafa1, Eman, M. Rashad2 and Salam, S. Teleb2

1Department of Entomology, Faculty of Science, Ain Shams University, Egypt.2Department of Zoology, Faculty of Science, Zagazig University, Egypt.

salamteleb@gmail.com

Abstract: Studies were undertaken to investigate haemolymph amino acidsalterations by amino acid analyzer in the 5th nymphal instar and adultSchistocerca gregaria in preoviposition and oviposition periods before and afterpyridalyl treatment. The concentrations of these amino acids in both controland treated haemolymph samples of the 5th nymphal instars showed a wide rangeof variations. The pattern of amino acids showed an obvious increase in thehaemolymph of the nymph after treatment with pyridalyl. Methionine andtryptophan were completely lost in untreated and treated nymphs.The pattern ofsome amino acids in 1-day old adult exhibited obvious increase in thehaemolymph after treatment with pyridalyl, while proline, threonine, glycineand arginine showed a decrease in their concentrations after treatment. On theother hand, most of the amino acids exhibited an obvious decrease in thehaemolymph of the 10 days old adult females after treatment with pyridalyl.Also, the amino acids in the haemolymph of the 18 days old adult femalesexhibited an obvious decrease after treatment with pyridalyl except threonine,serine, phenylalanine, glutamic acid and alanine.[Zahia, K. Mostafa, Eman, M. Rashad and Salam, S. Teleb, Haemolymph amino acids alterations in pyridalyl treated desert locust, Schistocerca gregaria in relation to age.] Life Science Journal 2012; 9(x):-]. (ISSN: 1097-8135). http://www.lifesciencesite.com.

Keywords: Schistocerca gregaria, haemolymph, amino acids, pyridalyl.

1. Introduction Pyridalyl (S-1812; 2, 6-dichloro-4-(3, 3-dichloroallyloxy)phenyl 3- (5-(trifluoromethyl)-2-pyridyloxyl propyl ether], is anovel insecticide that has aphenoxy-pyridaloxy derivativestructure, introduced by SumitomoChemical Co, Ltd. The compound wasreported effective on the pests oforder Lepidoptera and Thysanopteraon cotton and vegetables, withoutany phytotoxicity (Sakamoto et al., 2003). Its efficacy

was also reported against populations

of tobacco budworm, Heliothis virescens,cotton bollworm, Helicoverpa zea (Johnsonet al., 2000) and Plutella xylostella (Umedaand Strickland, 1999) which areresistant to various currently usedinsecticides. Pyridalyl may have a different

mode of action from other existinginsecticides. Pyridalyl is also lessharmful than existing insecticides tovarious beneficial arthropods, so itshould provide an important tool inIPM and insecticidal managementprogrammes for the control oflepidopterous and thysanopterous

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pests. However, no direct proof hasbeen found on the biochemicalmechanism of pyridalyl has not beenclarified to date. The haemolymph, in mostinsects, is a greenish yellowishfluid containing a large number ofcells or haemocytes and variousorganic and inorganic constituents.The insect haemolymph serves as abathing medium for various tissuesand organs as they lack anepithelial lining of a true coelom.Therefore, the haemolymph forms themeeting place of both the rawmaterials required and the productsof various physiological activitiesof the insect body. Since thehaemolymph is not directly connectedwith the external environment anychange in it can be taken as ameasure of the physiological stateof the internal environment of theintact animal. Analysis of thehaemolymph, therefore, may provideone of the most reliable data whichcan be used as an index of thephysiological activity (Buck, 1953;Wyatt, 1961). The insect haemolymphis noted for its high titer of freeamino acids (Chen, 1962). The mostimportant functions of amino acids,the building blocks of the proteinsthat are derived from the insectdiet, include the synthesis ofstructural proteins of theintegument and the synthesis ofhormones and enzymes thatparticipate in the synthesis nucleicacids (Klowden, 2007). Amino acidsare also required by insects fortransport and storage, and asreceptor molecules. In addition,some amino acids are involved inmorphogenesis (Chapman, 2002). Thepattern of haemolymph free amino

acids can be modified bydevelopment, oogenesis, feeding,cuticular tanning, silk production,or flight activity (Blum, 1985).Therefore, the present work aims tostudy the haemolymph amino acidsalterations in the 5th nymphalinstar and adult of Schistocercagregaria in preoviposition andoviposition periods before and afterpyridalyl treatments.

2. Material and Methods Insects:

The colony of Schistocerca gregaria(S. gregaria) was maintained from AntiLocust Research Center Dokki –Giza,Egypt. The insects were maintainedin the laboratory under crowdedconditions at 32±2ºC and 65-80% RHand reared according to Hunter-Jones(1961) and Hassanein (1965).

Administration of the insecticide: Groups of 50 hoppers of 1- day

old 4th nymphal instars were treatedwith 500 p.p.m. of pyridalyl® whichis a novel insecticide that has aphenoxy-pyridaloxy derivativestructure (S-1812; 2, 6-dichloro-4-(3, 3-dichloroallyloxy) phenyl 3-(5-(trifluoromethyl)-2-pyridyloxylpropyl ether). The concentration waschosen depending on some preliminarytrials carried out on the presentinsect species. Feeding techniquewas applied using fresh cleanlettuce (Lactuca sativa) after dippingfor 3 minutes. Feeding on treatedfood plant was allowed for 24 hrs.Controls were fed for 24 hrs onlettuce leaves dipped in tap waterfor 3 minutes and dried in open air.After feeding for 24 hrs, thetreated nymphs were daily fed onuntreated fresh lettuce and kept

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under the same laboratoryconditions.

Sample preparation for the assay of total amino acids in the haemolymph:

Haemolymph from the 5-days old5th nymphal instars, 1-day old adultfemales, 10-days old adult femalesand 18- days old adult females wasdrawn by a fine capillaries througha puncture in the neck membrane,pooled into Eppendorff tubes, storedin ice, containing few milligrams ofphenylthiourea to prevent tanning ordarkening and then diluted fivetimes with saline solution 0.7%. Thehaemolymph samples were thencentrifuged at 2000 r.p.m. for 5min, and only the supernatantfractions were used for assaydirectly or frozen until used.

Analysis of amino acids in the haemolymph with amino acid analyzer:

The haemolymph from the 5-daysold 5th nymphal instars, 1-day oldadult females, 10-days old adultfemales and 18-days old adultfemales of S. gregaria treated anduntreated were collected and kept at-20˚C. Amino acid analysis wascarried out by amino acid analyzer.The samples were hydrolyzed insealed, evacuated ampoules in anoven at 110˚C for 16 hrs. Theextraction and analysis wereperformed in Cairo UniversityResearch Park (CURP) at Amino AcidsLab. according to the methoddescribed by Rashad and Abdel Zaher(2008).

3. Results1. Total amino acids in the

haemolymph of the 5th nymphalinstar:

The total amino acid profile ofhaemolymph of S. gregaria nymph wasanalyzed using the Amino AcidAnalyzer and the results arepresented in figure (1). The analysisdetected the amino acids in controland treated nymphs. Comparison ofthe concentrations of these aminoacids in both control and treatedhaemolymph samples showed a widerange of variations. It appears thatthe pattern of amino acids showed anobvious increase in the haemolymphof the nymph after treatment withpyridalyl. The relative abundance oftotal amino acids in the haemolymphof the control nymph was as follows:glycine > glutamic acid > asparticacid > leucine > alanine > valine >lysine > serine > tyrosine >histidine > threonine > arginine >isoleucine > phenylalanine. Theconcentration of proline was muchlower.

The relative abundance of totalamino acid profile in pyridalyltreated nymph was as follows:glutamic acid > glycine > asparticacid > leucine > valine > alanine >tyrosine > lysine > histidine >isoleucine > arginine >phenylalanine > threonine > serine.Proline showed no obviousquantitative changes in thehaemolymph of the nymph aftertreatment with pyridalyl. Methionineand tryptophan were completely lostin untreated and treated nymphs. Thedifference between untreated andtreated nymphs showed a significantincrease in individual free aminoacids except proline.

2- Total amino acids in thehaemolymph of the adult S. gregaria inrelation to age:

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2.1. Total amino acids of 1day oldadult:

The data presented in figure(2) shows that the 15 amino acidswere also detected in control andtreated 1day old adult of S. gregaria. It appears that the patternof some amino acids exhibited anobvious decrease in the haemolymphof the adult after treatment withpyridalyl; these amino acids wereproline, glycine and arginine. On

the other hand, some amino acidsshowed an obvious increase in thehaemolymph of the adult aftertreatment with pyridalyl, e.g.aspartic acid, leucine, alanine,valine, lysine, histidine,phenylalanine, tyrosine, isoleucine,serine, and glutamic acid. Threonineshowed no obvious quantitativechanges in the haemolymph of theadult after treatment withpyridalyl.

Figure (1): Effect of pyridalyl on the haemolymph amino acid (g/100ml) in thecontrol and treated nymphs of S. gregaria.

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Figure (2):Effect of pyridalyl on the haemolymph amino acid (g/100ml) in thecontrol and treated 1day old adult of S. gregaria.

2.2. Total amino acids of 10 days old adult:

The data presented in figure(3) shows that most of the aminoacids exhibited an obvious decreasein the haemolymph of the 10 days oldadult females after treatment withpyridalyl; these amino acids wereglycine, glutamic acid, aspartic

acid, leucine, valine, histidine,lysine, tyrosine, isoleucine, serineand threonine. Alanine,phenylalanine and arginine showed noobvious quantitative changes in thehaemolymph of the adult aftertreatment with pyridalyl. Prolineexhibited some increasing in treated10 days old adult females.

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Figure (3): Effect of pyridalyl on the haemolymph amino acid (g/100ml) in the control and treated 10 days old adult of Schistocerca gregaria.

2.3. Total amino acids of 18 days old adult:

Data presented in figure (4)shows that all the amino acidsexhibited an obvious decrease in thehaemolymph of the 18 days old adultfemales after treatment with

pyridalyl except threonine, serine,phenylalanine, glutamic acid andalanine. Threonine, serine andphenylalanine showed an obviousincrease after treatment whileglutamic acid and alanine werecompletely lost.

Figure (4):Effect of pyridalyl on the haemolymph amino acid (g/100ml) in thecontrol and treated 18 days old adult of S. gregaria.

Comparison between the pattern ofhaemolymph amino acids in the chosenexperimental stages showed that theconcentration of glycine was thehighest value in the normal 1-dayold adult (3.039 g/100ml) followedby a decrease in 10-days old adult(2.937 g/100ml) then 5th nymphalinstar(2.270 g/100ml) and 18-daysold adult(1.089 g/100ml) as shown intable (1). Glutamic acid showed noobvious change between the chosenstages except it’s highly increasein the 5th nymphal instar (11.339g/100ml) and it’s lost in 18-daysold adult after treatment withpyridalyl. Aspartic acid showed thelowest value in the normal 1-day old

adult (0.815 g/100ml) than the 5th

nymphal instar (0.931 g/100ml). Itsvalue was decreased in normal 10-days old adult (0.682 g/100ml) thenwas increased in18-days old adult(0.889 g/100ml). After treatmentwith pyridalyl, aspartic acid wasincreased in both 5th nymphal instar(8.260 g/100ml) and 1-day old adult(1.508 g/100ml) and decreased in 10-days old adult (0.563 g/100ml)followed by in18-days old adult(0.196 g/100ml). The other aminoacids were fluctuated as shown intable (1) between increasing anddecreasing after treatment withpyridalyl.

Table (1): Total amino acid profile of haemolymph in pyridalyl control andtreated 5th nymphal instar, 1-day old adult, 10-days old adult and 18-days old adult of Schistocerca gregaria.

18daysoldadulttreated

18daysoldadultcontrol

10daysoldadulttreated

10daysoldAdultcontrol

1dayoldadulttreated

1dayoldadultcontrol

5th

nymphtreated

5th

nymphcontrol

Amino acidsNo.

0.1960.8890.5630.6821.5080.8158.2600.931Asparticacid

1

0.2410.1990.2320.3140.3390.3462.4010.362Threonine20.2430.1860.3580.4030.4980.2822.2910.423Serine3-1.5771.3031.5771.5951.42711.33

91.728Glutamic

acid4

0.0530.1170.1080.0640.010.0820.1770.038Proline51.0331.0892.6972.9371.7373.0399.6522.270Glycine6-0.2530.5840.6010.8600.3895.4540.630Alanine7

0.0390.3350.1500.3380.8760.4886.5100.543Valine80.0550.1380.0650.1510.5080.2062.9800.255Isoleucine90.0980.5960.1760.4391.2230.5587.7560.752Leucine100.1360.2290.1170.1970.4030.1854.6090.390Tyrosine110.1750.0370.0320.0400.4230.0522.5520.116Phenylalani

ne12

0.1730.2680.0910.3250.5450.3543.1960.377Histidine130.0580.4150.1200.2670.6760.3664.7040.504Lysine140.2980.4390.1900.1930.3410.5142.5920.327Arginine15

4. DiscussionAnalysis of haemolymph of 5th

nymphal instar, 1day old adult, 10days old adult and 18 days old adultof S. gregaria revealed the presenceof 15 amino acids, the concentrationof which in normal and treatedsamples showed a wide range ofvariation. All amino acids showed anobvious increase in the haemolymphof the 5th nymphal instar aftertreatment with pyridalyl. Thedisappearance of methionine as anessential amino acid may be due toits consumption in the methylatingintermediary pathways (Boctor,1978). The fact that arginineincreased after treatment wasrelated to its proposed function inthe metabolic process in the healthynymph or to its accumulation asguanidine derivative (Pant &Agrawal, 1964). In the presentstudy, increasing the amino acids inthe treated 5th nymphal instar of

S. gregaria may be due to theincrease representing thecompensation for the loss ofchlorides and other inorganic ionswhich fall during starvation whenosmotic pressure falls(Wigglesworth, 1972) as well as itmight be interpreted as being due tothe inhibition of protein formation(Bakr et al., 1991). They added thatthe variations in quantity of thefree amino acids content mayinterfere in the transcript of DNAduring the process of proteinsynthesis which may be the reason ofthe observed abnormalities.

The high levels of amino acidsin treated insects may cause a greatdisturbance in biochemicalactivities as protein metabolism aswell as in the regulation of osmotic

pressure (Enan, 2004). Blum (1985)reported that the use of amino acidsfor osmoregulation may at times bepassive process in which all aminoacids increase or decreaseproportionally, but the change inspecific amino acid titers in someinsects indicates active regulatedprocesses.

The remarkable increase ofglycine, glutamic acids and asparticacid in the chosen stages indicatedthat these are the predominant aminoacids of S. gregaria. Blum (1985) statedthat glutamate, proline and glycineare the predominant amino acids inmost insect haemolymph. In thepresent study, proline was presentin low concentration, this may bedue to it does not used as a sourceof energy. Chapman (2002) explainedthat locusts depend mainly on fat asa fuels providing energy for flightbut use carbohydrates during shortflight and the early stages ofsustained flight. Fat is moresuitable than carbohydrates as areserve for insects that make longflight because it produces twice asmuch energy per unit weight. Thelower concentration of proline andhighest concentration of glutamicacid and alanine may be due to theconversion of proline to glutamateas mentioned by Klowden (2007). Hestated that the proline is convertedto glutamate by the enzyme prolinedehydrogenase, which is activated byhigh level of pyruvate, thetransamination of glutamate withpyruvate produce the alanine. Ray(1964) stated that the loss of freeproline was balanced by an increasein free alanine and glutamine.

On the other hand, the massiveproduction of certain kinds of

proteins alters the haemolymph aminoacids pattern (Blum, 1985). Inspecies like locusts andcockroaches, with a discontinuousoviposition rate, there is a cyclicrise and fall of haemolymphvitellogenins concentrationcorresponding to the extent ofovarial maturation of eggs (Blum,1985). These statements of Blumexplain the increasing of glycine in1-day old adult followed bydecreasing in10-days old adult thenincreased again at 18-days oldadult. The 1-day old adultrepresents the beginning of thefirst cycle followed by the end at10-days old adult. The beginning ofthe second cycle was represented in18-days old adult.

Collett (1976) recorded thatglycine was active in the synthesisof several proteins. Although theresults from this study did notconclusively show that pyridalylinterferes with cell function, itwould be worthwhile to investigateits effect on cell function in afuture study.

Corresponding authorSalam, S. TelebDepartment of Zoology, Faculty of Science, Zagazig University, Egypt.salamteleb@gmail.com

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