Effects of Starvation, Dietary Regimes, and Temperature Stress on Hemocyte Profiles and Phenoloxidase Activity in Larvae of Tuta absoluta Meyrick (Lepidoptera: Gelechiidae)

Articles in Press

Document Type : Original Research

Authors
J. Karimian
M. Ajam Hassani
Department of Plant Protection, Faculty of Agriculture, Shahrood University of Technology
Abstract
This study examines changes in hemocyte profiles and phenoloxidase activity in larvae of Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) under conditions of starvation, dietary variation, and thermal stress in laboratory settings. T. absoluta-infected tomato fruits were collected from fields and transported to laboratory, where larvae were extracted from fruits after two generations of rearing, Hemolymph was extracted with a capillary tube and placed on a slide. Hemocytes were identified through Giemsa staining and observed under light microscopy at 40× magnification. Starvation stress was induced for 12 and 24 hours while the control group remained unstressed. Hemocyte counts were determined using a hemocytometer under light microscopy at 40× magnification. Starved larvae exhibited significantly reduced total hemocyte counts, plasmatocytes, and granulocytes compared to the control group. Larvae reared on eight tomato varieties (Superchef, Basimo, Hartiva, Berantta, Breivio, Gs15, 1012, and 8320) displayed variable hemocyte densities, with the highest counts observed in those fed on Superchef and Gs15 cultivars. For thermal stress experiments, third- and fourth-instar larvae were exposed to 28°C and 4°C for 12 and 24 hours. Control groups for third and fourth instar larvae maintained at 25 ± 1°C. In total hemocyte and granulocyte densities were significantly reduced across all thermal treatments compared to controls. Plasmatocyte counts in third-instar larvae subjected to 12 hours of heat stress (327.5±18 cell/mm3 hemolymph) and cold stress (320±34.3 cell/mm3 hemolymph) were higher than those in control (294.3±23.3 cell/mm3 hemolymph). Phenoloxidase activity exhibited a direct correlation with hemocyte alterations across all experimental conditions. This study provides a foundation for further investigations into the pest's physiological defense mechanisms.

Keywords

Subjects

Ajamhassani, M. 2021. Hemocyte changes of larvae of the beet moth, Scrobipalpa ocellatella (Lepidoptera: Gelechiidae) affected by thermal stress. JESI, 41(1):101–103. (In Persian with English summary).
Ajamhassani, M., Ebrahimizadeh, Z., Abdos, F., Ahangi rashti, B. 2023. Different pistachio cultivars impair hemocyte frequencies in diapausing and nondiapausing larvae of pistachio seed chalcid, Megastigmus pistaciae (Hymenoptera: Torymidae). JESI, 43(4): 345-358.
Ajamhassani, M., El Aaalaoui, M., Valizadeh, B. 2025. Study on hemogram and the effect of thermal stress on hemocytes and development in Dacus ciliatus (Diptera: Tephritidae). JAST., 27(6).
Ayres, B. S., Varela Junior, A. S., Corcini, C. D., Lopes, E. M., Nery, L. E., Maciel, F. E., 2024. Effects of high temperature and LPS injections on the hemocytes of the crab Neohelice granulate. J Inv Pathol., 205, 108144.
Banville, N., Browne, N., Kavanagh, K. 2012. Effect of nutrient deprivation on the susceptibility of Galleria mellonella larvae to infection. Virulence., 3: 497-503.
Beckage, N.E. 2007. Insect immunology, 1st edition. Amsterdam, The Netherlands: Apple Academic Press.
Blanco, L.A.A., Crispim, J.S., Fernandes, K.M., de Oliveira, L.L., Pereira, M.F., Bazzolli, D.M.S. & Martins, G.F. 2017. Differential cellular immune response of Galleria mellonella to Actinobacillus pleuropneumoniae. Cell Tissue Res., 370(1): 153-168.
Bruno, D., Montali, A., Gariboldi, M., Wronska, A., Kaczmarek, A., Mohamed, A., Tian, L., Casartelli, M. and Tettamanti, G. 2022. Morphofunctional characterization of hemocytes in black soldier fly larvae. Insect Sci., 1-21.
Campero, M., Block, M., Ollevier, F. and Stocks, R. 2008. Correcting the short-term effect of food deprivation in a damselfly: mechanisms and costs. J. Anim. Ecol., 77: 66–73.
Carper, A.L., Enger, M.C. & Bowers, M.D. 2019. Host plant effects on immune response across development of a specialist caterpillar. Front. Ecol. Evol., 7, 208.
Castillo, J.C., Robertson, A.E. &Strand, M.R. 2006. Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti. Insect Biochem. Mol. Biol, 36, 891–903.
Catalán, T.P., Wozniak, A., Niemeyer, H.M., Kalergis, A.M. and Bozinovic, F., 2012. Interplay between thermal and immune ecology: effect of environmental temperature on insect immune response and energetic costs after an immune challenge. J. Insect Physiol. 58: 310- 317.
Cerenius, L., Soderhall, K., 2004. The prophenoloxidase-activating system in invertebrates. Immunol. Rev. 198, 116–126.
Chaui–Berlinck, J.G., Navas, C.A., Monteiro, L.H.A., Bicudo, J.E.P.W., 2004. Temperature effects on a whole metabolic reaction cannot be inferred from its components. Proc. R. Soc. Lond. B Biol. Sci. 271, 1415–1419.
Chown, S. L. and Nicolson, S. 2004. Insect Physiological Ecology: Mechanisms and Patterns Vol. 254 (Oxford University Press, Oxford).
Coqueret, V., Le Bot, J., Larbat, R., Desneux, N., Robin, C. and Adamowicz, S. 2017. Nitrogen Nutrition of Tomato Plant Alters Leafminer Dietary Intake Dynamics. J. Insect Physiol., 99: 130–138.
Desneux, N., Luna, M.G., Guillemaud, T. and Urbaneja, A. 2011. The invasive South American tomato pinworm, Tuta absoluta, continues to spread in Afro-Eurasia and beyond: the new threat to tomato world production. J Pest Sci., 84: 403-408.
Dyar, H. C., 1890. The number of molts of lepidopterous larvae. Psyche. 5: 420-422.
Ehrlich, R.L., Zuk, M., 2019. The role of sex and temperature in melanin-based immune function. Can. J. Zool. 97, 825–832.
Ferracini, C., Ingegno, B.L., Navone, P., Ferrari, E., Mosti, M., Tavella, L. and Alma, A. 2012. Adaptation of indigenous larval parasitoids to Tuta absoluta (Lepidoptera: Gelechiidae) in Italy. J. Econ. Entomol., 105: 1311–1319.
Ghaderi, S., Fathipour, Y. and Asgari, S. 2017. Susceptibility of Seven Selected Tomato Cultivars to Tuta absoluta (Lepidoptera: Gelechiidae): Implications for Its Management. J. Econ. Entomol., 110: 421–442.
Gharekhani, G.H. and Salek-Ebrahimi, H. 2014. Evaluating the damage of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) on some cultivars of tomato under greenhouse condition. Arch. Phytopathol. Plant Prot., 47: 429-436.
Ghasemi, V., Moharramipour, S., and Jalali Sendi, J., 2013. Circulating hemocytes of Mediterranean flour moth, Ephestia kuehniella Zell (Lep: Pyralidae) and their response to thermal stress. ISJ., 10:128-140.
Gogoi, R., Kalita, K., Dey, D. & Ahmed, R. 2023. Haemocytes and their total count in 5th instar larvae of Eri silkworm, Samia cynthia ricini Boisduval (Lepidoptera: Saturniidae) reared on two host plants (castor and tapioca). Int. J. Entomol.Res., 7(11): 1-6.
Goodhead, L., and MacMillan, F. 2017. Measuring osmosis and hemolysis of red blood cells. Advances in Physiology Education., 41(2): 298-305.
Gourgoulianni, N., Schafer, M. A., Kapun, M., Busso, J. P., Blanckenhorn, W.U. 2023. Temperature-dependent melanism and phenoloxidase activity in the dimorphic sepsid fly Sepsis thoracica. J. Therm. Biol. 112 (103473)
Herren, P., Hesketh, H., Dunn, A. M. and Meyling, N. V. 2023. Heat stress has immediate and persistent effects on immunity and development of Tenebrio molitor. J. Insects Food Feed., 1-19.
Kang, K.D., Kamita, S.G., Suzuki, K. and Seong, S. 2011. Effect of starvation upon baculovirus replication in larval Bombyx mori and Heliothis virescens. J. Inv. Path. 106: 205-210.
Kellermann, V., van Heerwaarden, B., Sgro, C. M. and Hofmann, A. A. 2009. Fundamental evolutionary limits in ecological traits drive Drosophila species distributions. Sci., 325: 1244–1246.
Kholghahmadi M, Karimi-Malati A, Jalali Sendi J. 2025. Larval instar-dependent hemocytes in specialist herbivore Cydalima perspectalis fed on Buxus hyrcana and Buxus microphylla. Physiol. Entomol. 1-13.
Klepsatel, P., Girish, T.N., Dircksen, H., Gáliková, M., 2019. Reproductive fitness of 617 Drosophila is maximised by optimal developmental temperature. J. Exp. Biol. 222: 618 jeb202184.
Krechemer, F.D.S. and Foerster, L.M. 2015. Tuta absoluta (Lepidoptera: Gelechiidae): thermal requirements and effect of temperature on development, survival, reproduction and longevity. Eur. J. Entomol., 112: 658–663.
Laughton, A.M., O’Connor, C.O., Knell, R.J., 2017. Responses to a warming world: integrating life history, immune investment, and pathogen resistance in a model insect species. Ecol. Evol. 7, 9699–9710.
Lavine, M. D., and Strand, M. R. 2002. Insect hemocytes and their role in immunity. Insect biochem.moL. biol., 32(10): 1295-1309.
Littlefair, J., Knell, R. 2016. Within- and trans-generational effects of variation in dietary macronutrient content on life-history traits in the moth Plodia interpunctella. Plos one. 11: 1-16.
Lord, J. C. 2010. Dietary stress increases the susceptibility of Tribolium castaneum to Beauveria bassiana. J. Econ Entomol. 103: 1542-1546.
Lubawy, J. and Sticinska, M. 2020. Characterization of Gromphadorhina coquereliana hemolymph under cold stress. Sci. Rep., 10:12076.
Maingi, F. M., Akutse, K. S., Ajene, I. J., Omolo, K. M., Khamis, F. M. 2023. Immunological responses and gut microbial shifts in Phthorimaea absoluta exposed to Metarhizium anisopliae isolates under different temperature regimes. Front. Microbiol., 1-16.
Manjula, P., Lalitha, K., Vengateswari, G., Patil, J., Senthil, S.N. and Shivakumar, M.S. 2020. Effect of Manihot esculenta (Crantz) leaf extracts on antioxidant and immune system of Spodoptera litura (Lepidoptera: Noctuidae). Biocata and Agri Biotech., 23: 1-9.
Mason, A. P., Smilanich, A. M. and Singer, M. S., 2014. Reduced consumption of protein-rich foods follows immune challenge in a polyphagous caterpillar. J. Exp. Biol. 217: 2250-2260.
Mirhosseini, M.A., Sohrabi, F., Michaud, J. P., Rezaei, M. 2022. Suitability of Five Tomato Cultivars for Tuta absoluta (Lepidoptera: Gelechiidae): Resistance Is Associated with a High Density of Glandular Trichomes. J. Agr. Sci. Tech., 24(4): 837-846
Mowlds, P., Kavanagh, K. 2008. Effect of pre-incubation temperature on susceptibility of Galleria mellonella larvae to infection by Candida albicans. Mycopathologia., 165: 5-12.
Myers, J.H., Cory, J.S., Ericsson, J.D. and Tseng, M.L. 2011. The effect of food limitation on immunity factors and disease resistance in the western tent caterpillar. Oecologia., 167: 647-655.
Nakahara, Y., Kanamori, Y., Kiuchi, M. and Kamimura, M. 2003. In vitro studies of hematopoiesis in the silkworm: Cell proliferation in and hemocyte discharge from the hematopoietic organ. J Insect Physiol., 49: 907-916.
Nyamukondiwa, C. and Terblanche, J.S., 2010. Within-generation variation of critical thermal limits in adult Mediterranean and Natal fruit flies Ceratitis capitata and Ceratitis rosa: thermal history affects short-term responses to temperature. Physiol. Entomol., 35: 255–264.
Pandey, J. P., Tiwari, R. K. and Kumar, D. 2008. Temperature and Ganglionectomy Stresses Affect Haemocyte Counts in Plain Tiger Butterfly, Danaus chrysippus L. (Lepidoptera: Nymphalidae). J. Entomol., 5: 113-121.
Pourali, Z. and Ajamhassani, M. 2018. The effect of thermal stresses on the immune system of the potato tuber moth, Phthorimaea operculella (Lepidoptera: Gelechiidae). JESI., 37(4): supplementary, 515-525.
Roger, N., Michez, D., Wattiez, R., Sheridan, C. and Vanderplanck, M. 2017. Diet effects on bumblebee health. J. Insect Physiol., 96: 128-133.
Rostami, E., Madadi, H., Abbasipour, H., Allahyari, H. and Cuthbertson, A. G. S. 2017. Life Table Parameters of the Tomato Leaf Miner Tuta absoluta (Lepidoptera: Gelechiidae) on Different Tomato Cultivars. J. Appl. Entomol., 141: 88–96
Shapiro, M. 1979. Changes in hemocyte populations. In: Gupta A.P. (ed.), Insect hemocytes, Cambridge University Press, Cambridge, pp. 475-524.
Sinclair, B. J., Alvarado, L. E. C. and Ferguson, L. V. 2015. An invitation to measure insect cold tolerance: methods, approaches, and workfow. J. Term. Biol., 53: 180–197.
Siva-Jothy, M. T. and Thompson, J. J. 2002. Short-term nutrient deprivation affects immune function. Physiol. Entomol., 27(3): 206–212.
Stączek, S., Zdybicka-Barabas, A., Wiater, A., Pleszczyńska, M. and Cytryńska, M. 2020. Activation of cellular immune response in insect model host Galleria mellonella by fungal α-1, 3-glucan. Pathog Dis., 78(9): ftaa062
Strand, M. R. 2008. The insect cellular immune response. Insect Sci., 15: 1- 14
Triggs, A., Knell, R.J. 2011. Intractions between environmental variables determine immunity in the indian meal moth Plodia interpunctella. J Anim. Ecol., 1-9.
Vengateswari, G., Arunthirumeri, M. and Shivakumar, M. S. 2020. Effect of food plants on Spodoptera litura (Lepidoptera: Noctuidae) larvae immune and antioxidant properties in response to Bacillus thuringiensis infection. Toxicol. Rep., 7: 1428-1437.
Veyrat, N., Robert, C.A.M., Turlings, T.C.J. & Erb, M. 2016. Herbivore intoxication as a potential primary function of an inducible volatile plant signal. J. Ecol., 104, 591–600.
Vogelweith, F., Moret, Y., Monceau, K., Thieri, D. and Moreau, J. 2016. The relative abundance. of hemocyte types in a polyphagous moth larva depends on diet. J. Insect physiol., 88: 33-39.
Yeager, J. F. 1945. The blood picture of the Southern armyworm (Prodenia eridamin). J. Agric. Res., 71: 1–40.
Zhong, K., Liu, Z.-C., Wang, J.-L., and Liu, X.-S. 2017. The Entomopathogenic fungus Nomuraea rileyi impairs cellular immunity of its host Helicoverpa armigera. Arch. Insect. Biochem. Physiol. 96:21402.
Zhu, Q., Yuan, H.Y., Yao, J., Liu, Y., Tao, L. and Huang, Q. 2012. Effects of sublethal concentrations of the chitin synthesis inhibitor, hexaflumuron, on the development and hemolymph physiology of the cutworm, Spodoptera litura. J Insect Sci., 12: 1- 13.
Journal of Agricultural Science and Technology

Articles in Press, Accepted Manuscript
Available Online from 01 January 2024