ACTA ENTOMOLOGICA SLOVENICA
LJUBLJANA, DECEMBER 2019 Vol. 27, st. 2: 109-116
FOOD PLANT STERILIZATION AND ITS IMPACTS ON HEMOCYTE CELLS AND ACTIVITIES OF CARBOHYDRASES IN THE MIDGUT OF ZONOCERUS VARIEGATUS (L.)
Ademolu K.O.1*, IDOWU A.B.1, Olabode O.A.1, Joda A.O.2 and Adelabu A.B.3
department of Pure and Applied Zoology, Federal University of Agriculture, Abeokuta, Nigeria department of Crop Protection, Olabisi Onabanjo University, Ago-Iwoye, Nigeria. department of Microbiology, Chrisland University, Owode, Abeokuta, Nigeria. Corresponding Author: kennyademolu@yahoo.com
Abstract - Through feeding, phytophagous insects acquire their gut microbiota which assists in enzymes secretion and production of secondary metabolites. This study seeks to validate the role of microbes found on food plants by sterilizing cassava leaves eaten by Zonocerus variegatus and its impacts on hemocyte cells and the midgut car-bohydrases (amylase, cellulase and a-glucosidase) activities. The insects were divided into two groups: insects fed with sterilized cassava leaves and others fed with unster-ilized cassava leaves. T-test was used to analyze the results. Five (5) hemocytes cells (prohemocyte, plasmatocytes, granulocytes, sperulocyte and adipohemocyte) were detected in the insects' haemolymph with the granulocytes recording the highest frequencies. Zonocerus variegatus fed with unsterilized cassava leaves had significantly higher hemocyte cells than those that consumed sterilized leaves. Similarly, Z. variegatus that consumed unsterilized leaves had significantly higher carbohydrases activities than those whose feeds were sterilized. Activities of a-glucosidase were higher than other enzymes in the midgut of the experimental Z. variegatus. It can be concluded that removal of microorganisms on the food plants eaten by Zonocerus through sterilization affected its immune response and digestive physiology.
Key words: Cassava leaves, sterilization, hemocytes, midgut, carbohydrases, Zono-cerus
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Izvleček - STERILIZACIJA HRANILNIH RASTLIN IN NJEN VPLIV NA CELICE HEMOCITE IN AKTIVNOST KARBOHIDRAZ V SREDNJEM ČREVESU KOBILICE ZONOCERUS VARIEGATUS (L.)
S hranjenjem rastlinojede žuželke pridobijo svoje črevesne mikrobe, ki pomagajo pri izločanju encimov in produkciji sekundarnih presnovkov. V študiji smo skušali potrditi vlogo mikrobov, ki jih najdemo na hranilnih rastlinah, tako da smo sterilizirali liste kasave, ki jo jedo kobilice vrste Zonocerus variegatus in raziskati njihov vpliv na celice hemocite in aktivnost karbohidraz srednjega črevesa (amilaza, celulaza in a-glukozidaza). Žuželke smo razdelili v dve skupini: v eni so jedle sterilizirane liste kasave, v drugi nesterilizirane. Za analizo rezultatov smo uporabili t-test. 5 vrst he-mocitnih celic (prohemociti, plazmatociti, granulociti, sperulociti in adipohemociti) smo našli v žuželčji hemolimfi in granulociti so dosegli največje gostote. Kobilice Zonocerus variegatus, hranjene z nesteriliziranimi listi kasave, so imele občutno več hemocitnih celic kot tiste, ki so zaužile sterilizirane liste. Prav tako so imele tiste, ki so zaužile nesterilizirane liste, občutno višjo aktivnost karbohidraz od onih, katerih hrana je bila sterilizirana. Aktivnost a-glukozidaze je bila višja od drugih encimov srednjega črevesa poskusnih živali. Lahko zaključimo, da je odstranitev mikroorganizmov s hranilnih rastlin, ki jih jedo kobilice rodu Zonocerus s sterilizacijo vplivalo na njihov imunski odziv in prebavno fiziologijo.
Key words: listi kasave, sterilizacija, hemociti, srednje črevo, karbohidraze, Zono-cerus
Introduction
The variegated grasshopper, Zonocerus variegatus (L.) was accorded pest status in Nigeria in 1974 by Centre for Overseas Pest Regulation, due to its polyphagus nature, consuming both food and cash crops (Toye, 1982; COPR, 1974). Chiffaud and Mestre (1970) reported that Z. variegatus consumed more than 250 plants among which are: citrus, cocoa, banana, vegetables and coffee. Also in the neighboring country, Cameroon, Z. variegatus was ranked 3rd most economically detrimental insect pest of agriculture (Kekeunou, 2006).
Methods adopted for its control include - physical, cultural, chemical and integrated pest management (Toye, 1982). However, due to its numerous side effects chemical method has been discouraged and other methods are now combined for effective control. One method yet to be fully harnessed is the biological control which involves the use of natural enemies as pathogens to control pest (Toye, 1982).
The roles played by hemocytes in the insects include phagocytosis of microbes, encapsulation of foreign materials, coagulation of haemolymph and distribution of nutritive materials (Gupta, 1985). Idowu and Sonde (2004) observed that food types consumed by insects influenced the types and number of hemocyte cells.
The gut of polyphagus insects like Zonocerus harbors numerous microorganisms. Namely: bacteria and fungi (Chapman, 1990; Campbell, 1990). The colony forming unit (cfu) of these microbes increased as the size and age of the insect increased
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(Idowu and Edema, 2004). Among other things, the microbes synthesize digestive enzymes especially cellulase which cannot be synthesized by animals (Idowu et al, 2009). Alcohols, vitamins and minerals are other by-products of fermentation process anchored by these microbes (Chapman, 1990). These microorganisms are not vertically transmitted from mothers to their offspring in Zonocerus but through the food plant consumed (Ademolu and Idowu, 2011).
The roles played by microbes in the physiology of insects are numerous, however, their significant roles at micro level (enzymes and immune response) has not received full attention in literature. The study aims to investigate the role of microbes in digestion process and effects on immune response. The focus of this study is to evaluate the effect of food (leaves) sterilization on gut enzymes and hemocytes number in Z. variegatus.
Materials and methods
INSECT COLLECTION AND MAINTENANCE
Variegated grasshoppers (Z. variegatus) of various stages of development were collected from uncultivated farmland near the Health Centre of Federal University of Agriculture, Abeokuta, (FUNAAB) Nigeria early in the morning (6:30 am - 7:30 am). The insects were sorted by sex and later instars (5th and 6th instars) were maintained on fresh cassava (Manihot esculenta) leaves until they reached the adult stage. Male adults of 3 days old were recruited for this study.
EXPERIMENTAL PROCEDURE
Forty (40) male adults Z. variegatus were separated into two groups (A and B) of twenty individuals each. Group A were fed with sterilized cassava leaves while insects in group B were fed with unsterilized cassava leaves. The two groups of insect were fed ad-libitum for 6 weeks.
The sterilization of the cassava leaves was done following procedures described by Rutala et al. (2008) and Ademolu et al. (2015).
HEMOCYTE ESTIMATION
Hemocytes estimation was done by making a thin smear of the insect haemolymph, air dried and hemocyte cells were detected with drops of Leishman's stain (Gupta, 1979). Freshly prepared slides were used for identifying cell types under microscope (x 400). Estimates of cell counts were made by counting number of different cells in three different fields of view (Jibir,1981). Quantification was performed in triplicate for each animal.
GUT PREPARATION
Three individual insects per group were frozen in a deep freezer for 30 minutes after which their guts were dissected out and the midguts were carefully removed and homogenized in 0.05M KCl and centrifuged at 500 rpm for 30 minutes at 5 0C. The enzymes extract was decanted into bottles and kept for enzyme analysis.
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GUT ENZYMES ANALYSIS
The activities of carbohydrases (a-glucosidase, amylase and cellulase) in the midgut of Z. variegatus were found out following methods described by Adedire and Balogun (1995). The enzymes activities were determined quantitatively by using Dinitrosalicyclic Acid Reagent (DNSA). The amount of reducing sugar produced at the end of incubation period was estimated spectrophotometrically at 550nm. The experiment was replicated three times.
STATISTICAL ANALYSIS
Data collected from the experiments were analyzed using t-test (t = 0.05).
Results
The number and types of hemocytes in the Zonocerus fed sterilized cassava leaves are presented in Table 1. Five (5) different types of hemocyte cells were detected in the insect haemolymph with Granulocytes representing the highest number of cells. Also, insects fed unsterilized cassava leaves had significantly higher number of he-mocyte cells than those fed sterilized cassava leaves.
Table 1: Estimate of hemocyte cells in Zonocerus variegatus (L) fed cassava leaves (x105 per ml of haemolymph)
HEMOCYTE CELLS	INSECTS FED STERILIZED LEAVES	INSECTS FED UNSTERILIZED LEAVES
Prohemocyte	8	10
Plasmatocyte	7b	12a
Granulocyte	10b	15a
Oenocytoid	0	0
Sperulocyte	3b	10a
Adipohemocyte	7b	11a
Total	35	58
*Means values in the same row having different superscript are significantly different at (p<0.05)
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The activities of carbohydrases in the midgut of Zonocerus fed sterilized cassava leaves are shown in Table 2. Insects fed unsterilized leaves had significantly higher cellulase and a-glucosidase activities than those fed sterilized leaves. The highest enzymatic activity in the midgut was detected for a-glucosidase, while amylase activity was the lowest.
Table 2: Carbohydrases activities (Abs/min) in the midgut of Zonocerus variegatus fed cassava leaves
S/N	SAMPLE	CELLULASE	AMYLASE	a - GLUCOSIDASE
1	Grasshopper fed sterilized leaves	0.678b	0.215	0.861b
2	Grasshopper fed unsterilized leaves	0.712a	0.221	0.868a
*Means values in the same column having different superscript are significantly different at (p<0.05).
Discussion
The number and types of hemocyte cells present in the haemolymph of Z. variegatus and in other insects generally varies with the physiological state of the insects (Mullin, 1985 and Idowu and Sonde, 2004). Zonocerus fed with unsterilized leaves had more hemocytes than their counterparts fed with sterilized leaves. Ademolu et al (2010) observed that the number of hemocytes increased as Z. variegatus increased in age (during embryonic development) and this is attributed to the increase in food consumption as the insect ages. The food plants consumed by Zonocerus harbor microorganisms which elicit or activate host defense system of the insect (Eslin and Provost, 1998). Hence, the lower hemocytes in the insect fed with sterilized leaves might be due to reduced/low microbial load on the leaves which elicit less of the insect defense system.
Five hemocyte types were detected in the haemolymph of the experimental insects namely prohemocyte, plasmatocyte, granulocyte, sperulocyte and adipohemocyte. Oenocytoids were not detected in the experimental insects and this runs contrary to the six (6) hemocyte cells earlier recorded in Z. variegatus by Idowu and Sonde (2004). Gouli et al. (2000) earlier observed that Oenocyloids were extremely rare and few numbers were also seen in Adelges tsugae, likely due to their insignificant role in fighting foreign agents. Similarly, Jone (1962) pointed out that Oenocytoids were either absent or questionably present in same orthopterans as they rapidly disintegrate outside the host. on the other hand, granulocytes were the most abundant, followed by plasmatocytes and prohematocytes. This might be connected to their
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functions. Both granulocytes and plasmatocytes are involved in phagocytosis of the foreign agents and also important in wound healing (Gupta, 1985).
One essential role of microbes in the gut of Zonocerus is to synthesize enzymes needed to break down food plants consumed by insects. Idowu et al (2009) reported that microbes inhabiting the gut of Zonocerus secrete cellulolytic enzymes used by the insects. The source of these microbes has been linked to the food plants (Ademolu and Idowu, 2011) and their removal by sterilization shown to influence the production of these enzymes. This explains the reduced carbohydrases activities in the midgut of Zonocerus fed sterilized leaves.
It is worthy to note that the two groups had the highest activities of a - glucosidase and lowest activities of amylase in the midgut. The main diet of the insect is cassava leaves which is a form of polysaccharide and requires high activity of amylase to break it down to disaccharides. However, digestion of polysaccharide begins at the foregut as the enzymes move backward from the site of secretion (midgut) to the fore gut (Hill and Orchard, 2015). Hence, higher a - glucosidase activities in the midgut is needed to hydrolyse the inflow of disacharides from the foregut. Detection of cel-lulase in the midgut of the insects suggests the presence of microbes as animals lack the ability to synthesize cellulase. Microorganisms live symbiotically in the gut of phytophagous insects and assist by synthesizing the enzymes required for the hydrolysis of cellulose portion of their diet.
Conclusion
This study suggests that food microbes present on the food plants of Z. variegatus aid digestion through enzymes secretion and elicit the insect's defense system.
References
Adedire C. O., Balogun R. A., 1995: Digestive enzymes and regional localization of proteolytic endopeptidases in alimentary canal of Kola nut weevil, Sophrorhi-nus insperatus Faust (Coleoptera: Curculionidea). Entomon 20: 183-189. Ademolu, K.O; Idowu, A.B., Olatunde, G.O., 2010: Hemocyte populations in Zonocerus variegatus (L) (Orthoptera:Pyrgomorhidae) during post embryonic development. Acta Entomologica Sinica 53(4): 470-473. Ademolu, K.O., Idowu, A.B., 2011: Occurrence and distribution of microflora in the gut regions of variegated Grasshopper Zonocerus variegatus (Orthoptera: Pyrgomorphidae) during development. Zoological Studies 50 (4): 409-415. Ademolu, K.O; Idowu, A.B; Olabode, O.A; Olalonye; Adelabu, A.B., Atayese, O., 2015: Effect of sterilized cassava leaves (Manihot esculenta) on growth and nutritional value of adult variegated grasshopper, Zonocerus variegatus (L). Biological and Environmental Sciences Journal for the Tropics 12(3): 117121.
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Campbell, B.C., 1990: On the role of microbial symbionts in herbivorous insects. In EA Bernays, ed. Insect-plant interactions. Boca Raton, FL: CRC Press, pp. 1-45.
Centre For Overseas Pest Research London (COPR), 1974: Control of Zonocerus variegatus (L) in Nigeria. Interim Report: 1972-1973. ODM Research Scheme R2727.
Chapman R.F., 1990: The Insect: Structure and Function. The English Language Book Society and Hodder and Stoughton, Great Britain. 968 pp.
Chiffaud J, J. Mestre, 1990: Le Croquet Puant Zonocerus variegatus (Linne, 1758): Essai de synthese bibliographique.Montpellier: CIRAD-PRIFAS, 140 pp.
Eslin, P., Provost, G., 1998: Variation in Drosophilia concentration of hemocytes associated with different ability to encapsulate Asobara tabida larvae parasitoid. Journal of Insect Physiology 42:549-555.
Gouli, V., Parker, B.L., Skinner, M., 2000: Hemocytes of Hemlock Wolly adelgid, Aldeges tsugae Annand (Homoptera: Adelgidae) and changes after exposure to low temperatures. Journal of Applied Entomology 124:201-206.
Gupta, A.P., 1985: Cellular elements in the haemolymph. In: Comprehensive Insect Physiology Vol. 3 (Kerkut, G.A. and Gilbert, L.T. eds.), pp. 401-451. Pergamon Press, Oxford, UK.
Hill S. R., Orchard I., 2005: Enzymes in the gut during starvation. Journal of Insect Physiology 51, 1-9.
Idowu, A.B., Sonde, O.A., 2004: The contribution of food plants to the growth and fecundity of Zonocerus variegatus (L.). Nigeria Journal of Entomology 21: 24-28.
Idowu A.B., Edema M.O., 2004: The microbial flora of the different gut regions of Zonocerus variegatus (L.) (Orthoptera: Pyrgomorphidae). Nigeria Journal of Plant Protection 20: 19-30.
Idowu, A.B., Edema, M.O., Oyedepo, M.T., 2009: Extra cellular enzyme production by microflora from the gut regions of the variegated grasshopper, Zonocerus variegatus (L.) (Orthoptera: Pyrgomorphidae). Int. J. Trop. Insect Sci. 29: 229235.
Jibir, J.J., 1981: Hemocytes and haemolymph characteristics in the African pest grasshopper, Zonocerus variegatus (L) (Acrididae: Pyrgomorphidae). M.Sc. thesis, University of Ibadan, Nigeria, 61pp.
Jones, J.C., 1962: Current concepts concerning insect hemocytes. Am. Zoologist. 2: 209-246
Kekeunou, S., Weise, S., Messi, J., Tamo, M., 2006: Farmers' perception on the importance of Zonocerus variegatus (L.) (Orthoptera: Pyrgomorphidae) in humid forest zone of Southern Cameroon. Journal of Ethnobiology and Eth-nomedicine 2, 1-12.
Mullins, D.E., 1985: Chemistry and physiology of the haemolymph. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology Vol.3 (Kerkut, G.A. and Gilbert, L.T. eds.), pp. 355-400. Pergamon Press, Oxford, UK.
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Rutala W.A, Weber D.J, Hicpac, 2008: Guideline for disinfection and sterilization in healthcare facilities, Atlanta, GA: uS Department of Health and Human Services, CDC; pp 38-39. Toye, S. A., 1982: Studies on the Biology of the Grasshopper pest Zonocerus varie-gatus (L.) (orthoptera: Pyrgomorphidae) in Nigeria. Insect Sci. application 3
(1): 1-7. Received / Prejeto: 18. 1. 2019
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