EKOLOGIJA. 2006. Nr.4. P. 34–39 © Lietuvos mokslų akademija, 2006 34 Ilona Bartninkaitė, Povilas Ivinskis, Jolanta Rimšaitė © Lietuvos mokslų akademijos leidykla, 2006

Scarce (Maculinea teleius) hemolymph studies in different populations of Lithuania

Ilona Bartninkaitė, hemolymph studies were conducted in different model populations of Lithuania. The analysis of the hemoplymph of , Povilas Ivinskis, and adults revealed hemolymph changes during the life cycle of the Scarce Large Blue. Proleucocytes were only found in the hemolymph of Jolanta Rimšaitė caterpillars, with no such type hemocytes in the hemolymph of adults. The amount of macronucleocytes also deceased in the hemolymph of Scarce Large Institute of Ecology of Blues as grew. Based on hemograms of adult Scarce Large Blues Vilnius University, collected in different areas we can conclude that their physiological state is Akademijos 2, LT-08412 Vilnius, good and the percentage ratio of different types of hemocytes changes insig- Lithuania nificantly in all study areas. E-mail: [email protected] Key words: hemolymph, Maculinea teleius, hemograms, hemocytes

INTRODUCTION The physiological state of an depends not on- ly upon the , but upon the time of insect deve- Biotic and abiotic environmental factors have a decisive lopment as well (Bartninkaitė, 1998, 2002), which can impact upon population numbers of insects. It is under be evidenced by hemolymph studies. their influence solely that insect fecundity, mortality and All this shows how versatile the importance of in- duration of their ontogenesis change. In determining the sect hemolymph is and why hemolymph studies are both functional regularities of insect populations, research into of practical and theoretical significance. their physiological state, which reveals the dynamics of The purpose of the present work was to study the internal processes within populations, is highly signifi- hemolymph of moths of the Maculinea teleius species cant. Hemolymph is one of the key indices of the in- during their ontogenesis and determine their physiologi- sect’s physiological state. Hemolymph, as a very labile cal state in different areas of the country to provide a tissue taking part in different live processes, most promp- basis for determining the viability of the population and tly responds to any stimuli. Even the slightest metabolic forecasting the status of this protected moth species in irregularity is initially reflected in insect hemolymph. The- Lithuania. refore, hemolymph as a key index of the physiological state is of paramount importance in studying the dyna- MATERIALS AND METHODS mics of insect population numbers (Sagdi, 1991; Bartnin- kaitė, Tekoriutė, 1994; Bartninkaitė, 1998). The percenta- Scarce Large Blue hemolymph smears were prepared ge ratio of different type form elements of hemolymph from caterpillars and adults collected in four places of allows a precise diagnosing of the insect’s physiological the country in 2005 by the accepted cytological met- state which provides information about the abundance and hods. The selected sites were meadows of Joniškis di- viability of the population (Jonaitis et al., 2000). strict near the Mūša (56°07’27.6”, 23°29’16.1”), Prie- The definition of an insect’s physiological state is kulė of Klaipėda district (55°31’40.2”, 21°22’47.7”), Ant- not only a basis for a precise forecasting of the insect vardė of Jurbarkas district (55°08’51”, 22°43’07”) and population numbers, but also could be an important in- Saviečiai of Kėdainiai district (55°09’02.9”, dex of environmental pollution (Solodovnikova et al., 23°59’00.7”). Hemolymph smears were fixed with me- 1990; Chkhoidze, 1991). Insect hemolymph studies can thyl alcohol, dyed using the Giemza–Romanovski me- also be used in insect because each type of thod and examined under an MBI-1 microscope. The species has an inherent species-specific percentage ratio classification of hemocytes, their percentage ratio as well and type-specific form and size (Sagdi, 1991; Bartnin- as the amount of mitotic divisions of proleucocytes and kaitė, 1995). macronucleocytes were estimated using the conventional Scarce Large Blue (Maculinea teleius) hemolymph studies in different populations of Lithuania 35 cytological methods, and the data obtained were treated changes. In the course of the development of caterpil- using the methods of variation statistics (Pristavko, 1971; lars, the number of proleucocytes in their hemolymph Lakin, 1980) and STATISTICA 6 software package. decreases on average from 15.3% in size III caterpillars Hemolymph smears from different size caterpillars to 10.4% in size V caterpillars (Table 1). Such differen- and adults were prepared for estimation of the dyna- ce in the number of proleucocytes in the hemolymph of mics of the hemolymph structure during the ontogenesis different size caterpillars is statistically reliable. As in of the Scarce Large Blue. The physiological state of the the course of development of caterpillars the number of Scarce Large Blue in separate areas was estimated ac- primary cells considerably decreases, the number of their cording to their hemograms (Bartninkaitė, 2002; Russo mitoses comes down, too. The average number of mi- J. et al, 2001). toses accounts for 21.6% of the total number of proleu- cocytes in the hemolymph of size III caterpillars and RESULTS AND DISCUSSION for merely 13.6% in the homolymph of size V caterpil- lars. Such difference is statistically reliable. The results We have detected five types of hemocytes in the hemo- show that when the body of insects ages, the process of lymph of the Scarce Large Blue during its ontogenesis. mitotic division of cells in their hemolymph becomes The percentage ratio of hemocytes was found to depend slower. upon the insect’s development stage. We found all five The number of macronucleocytes in the hemolymph types of form elements – proleucocytes, macronucleocy- also decreases as caterpillars grow. They average 40.7% tes, micronucleocytes, enocytoides and phagocytes – in in the hemolymph of caterpillars of size II and 38.4% the hemolymph of caterpillars and only three types of of size V (Table 1). The number of mitoses in macro- hemocytes – macronucleocytes, micronucleocytes and nucleocytes in the hemolymph of caterpillars is much phagocytes – in the hemolymph of adults. lower than that in proleucocytes. Depending upon the Proleucocytes are the smallest elements in the hemo- size, the number of mitoses in proleucocytes lymph of the Scarce Large Blue. A proleucocyte is an averages from 21.6 to 13.6%; in macronucleocytes, me- almost round cell (2.6–7.8 µk) with a large round nu- rely from 5.9 to 5.2%. Though the decrease in the cleus (1.1–6.5 µk) surrounded by a thin cytoplasm la- amount of macronucleocytes in the hemolymph of ca- yer (1–1.5µk). Proleucocytes are elementary cells from terpillars as they grow is not statistically reliable, the which other hemocytes develop. They can divide by decrease in the number of their mitoses is reliable. mitosis. Investigations of the hemolymph of different size ca- Macronucleocytes in the hemolymph of Scarce Large terpillars of the Scarce Large Blue have demonstrated Blue caterpillars are large (8.6–15.9 µk) round cells that as the body of an insect is ageing, not only the with a large nucleus (5.2–12.0 µk). The cytoplasm layer amount of young and little differentiated hemocytes in encircling the nucleus is considerably wider than that in its hemolymph decreases but also the mitotic activity of proleucocytes. One to three vacuoles can often be seen such hemocytes is slowing down. Such process in the in the cytoplasm of macronucleocytes. Macronucleocy- organism of an insect undoubtedly influences its life tes are young and little differential cells. Some of them duration. become mature in the course of ontogenesis and later Micronucleocytes are small (4.7–10.9 µk) nearly perform the function of phagocytosis, whereas others round cells with a rather small round nucleus (1.8– differentiate to other types of hemocytes. Macronucleo- 3.6µk) in the hemolymph of Scarce Large Blue cater- cytes can also divide by mitosis. They were found du- pillars. In the cytoplasm of micronucleocytes, several ring their mitotic division in the hemolymph of Scarce vacuoles and various dark dying insertions can be no- Large Blue caterpillars. ticed, which undoubtedly are nutrients carried by micro- As both proleucocytes and macronucleocytes are nucleocytes as mature trophic cells around the organism young and little differentiated cells, we have examined of the insect. Numerous authors indicate that how their amounts change in the hemolymph of cater- micronucleocytes perform the function of nutrient carri- pillars as they grow and how the frequency of mitoses ers in the organism and accumulate nutrients used

Table 1. Dynamics of proleucocytes and macronucleocytes and their mitoses in the hemolymph of different size Scarce Large Blue caterpillars

Caterpillar instar Proleucocytes Macronucleocytes

Amount of Number of Number of Amount of Number of Number of hemocytes % mitoses mitoses, % hemocytes % mitoses mitoses %

III 15.3 ± 0.88 3.3 ± 0.34 21.6 ± 1.02 40.7 ± 1.20 3.7 ± 0.67 8.94 ± 1.34 V 10.4 ± 1.03 1.4 ± 0.24 13.6 ± 2.28 38.4 ± 0.51 2.0 ± 0.32 5.20 ± 0.82 t 3.24 4.63 2.07 2.65 p value <0.05 <0.01 >0.05 <0.05 36 Ilona Bartninkaitė, Povilas Ivinskis, Jolanta Rimšaitė during insect (Sagdi, 1991; Bartninkaitė, smaller caterpillars, and for mere 8–14% in the hemo- 1995). lymph of larger ones, which could be explained by the Micronucleocytes differentiate from proleucocytes and transformation of proleucocytes into other types of macronucleocytes. They do not divide by mitosis as they hemocytes and a gradual slowing down of the forma- are mature trophic cells. tion of new proleucocytes up to a complete termination Enocytoides are among the largest cells (13.5–20.3 of their formation process in the course of development µk) in the hemolymph of Scarce Large Blue caterpil- of the insect. lars. They are nearly round in their shape with a small The amount of macronucleocytes in the hemolymph (3.1–7.8 µk) round nucleus in the middle. Enocytoides of the Scarce Large Blue is also decreasing as the in- are fully mature cells with no mitoses observed. Enocy- sect grows – from 40.7–38.4% in the hemolymph of toides perform the functions of elimination and secre- caterpillars to 2.5–3.7% in the hemolymph of adults tion in the organism (Sirotina, Chernaya, 1965). (Figs 1 and 2). As macronucleocytes are little differen- Phagocytes differ greatly in their shape from all other tiated cells, such a rapid decrease in their numbers du- hemocytes found in the hemolymph of the Scarce Large ring insect ontogenesis shows that macronucleocyte dif- Blue. They are spindle-shaped cells 18.2–26.5 µk long ferentiation into other type hemocytes and their matu- and 7.5–10.7 µk wide. The central part of phagocytes ring become more intensive in the organism of an in- contains a small oval nucleus (5.2–8.6 µk long and 2.6– sect as it grows. Mature macronucleocytes perform the 5.2 µk wide). It is noteworthy that phagocytes have no function of phagocytosis in the organism of an insect such long sprouts in the hemolymph of Scarce Large therefore, their abundance in the caterpillar stage shows Blue caterpillars compared with those found in the hemo- that macronucleocytes carry the greatest burden of pro- lymph of most caterpillars of the order , tective function in this particular stage wherefore their which sometimes reach 44

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Fig. 1. Scarce Large Blue hemograms during thel development of larvae 17% in the hemolymph of t s e s Scarce Large Blue (Maculinea teleius) hemolymph studies in different populations of Lithuania 37

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c Sites c y e t l e l s s Fig. 2. Scarge Large Blue hemograms in various sites mortality increases and consequently their numbers con- the insect, because it shows that hemocytes performing siderably decrease. the protective function in the organism of the insect In the hemolymph of Scarce Large Blue caterpillars, function actively and are able to eliminate different de- the number of micronucleocytes reaches 24–32 and in ad cells from the hemolymph. The number of dead cells adults 30–37 (Figs. 1 and 2). in the hemolymph of the Scarce Large Blue increases We have found very small numbers of enocytoides from 8.0–8.6% in the caterpillar stage (Fig. 1) to 19.3– in the hemolymph of caterpillars (Fig. 1) and have not 22.5% in the adult stage (Fig. 2). found them in the hemolymph of adult Scarce Large A comparison of Scarce Large Blue hemograms with Blues. Most authors reported analogous results as to the the hemograms of the other Lepidoptera insects shows hemolymph of different representatives of the order Le- that the physiological state of the Scarce Large Blue is pidoptera. rather good both in caterpillars and adults (Bauer et al., The amount of phagocytes during ontogenesis of the 1998, Jonaitis et al., 2000; Yamashita, Miyuki et al., Scarce Large Blue increases from 9.3–11.2% in the he- 2001; Bartninkaitė, 2002, 2003). High numbers of he- molymph of caterpillars up to 42.2–44.7% in the hemo- mocytes (macronucleocytes and phagocytes) specialized lymph of adults (Figs. 1 and 2). It could be explained for the protective function in the hemolymph of Scarce by a higher intensity of caterpillar feeding (as a conse- Large Blue caterpillars are implied by a small number quence of which the amounts of different substances of of dead cells – 8.0–8.6% on average (Figs. 1 and 2), alien origin getting into hemolymph become greater) and which evidences that Scarce Large Blue caterpillars are by a greater mortality of cells both in the hemolymph of good physiological state and will succeed in their and neighboring tissues (particularly in and adult development. Besides, the considerably greater number stages). Therefore, great numbers of phagocytes appear of macronucleocytes compared with phagocytes allows in the hemolymph whose function is to eliminate dead assuming that macronucleocytes perform the main pro- cells and harmful substances from the organism, so the tective function in the caterpillar stage, and as the in- number of phagocytes as cells performing the protective sect body is ageing, i.e. in the adult stage, the main function in the organism of an insect is indicative of protective function goes over to phagocytes, because the physiological state of the insect. their numbers in the hemolymph are tenfold the number The number of dead cells in the hemolymph is also of makronucleocytes at that time. an important index in determining the physiological sta- From the hemograms of Scarce Large Blue adults te of an insect. The smaller the number of dead cells in collected in different places (Fig. 2) we can conclude the hemolymph, the better is the physiological state of that their physiological state is good and similar in all 38 Ilona Bartninkaitė, Povilas Ivinskis, Jolanta Rimšaitė study areas and the percentage ratio of different type References hemocytes changes insignificantly. A statistically signifi- cant difference was detected only between the number 1.Bartninkaitė I. 1998. Dynamics of the hemolymph struc- of Enocytoides (t = 2.6, p value = 0.026) and dead cells ture of the nettle moth in various biotopes and its influ- (t = 2.26, p value 0.046) of imagos from Joniškis and ence on the trophic relations of the insect. Acta Zoologi- Saviečiai. A greater amount of dead cells was found in ca Lituanica. Vol. 8. P. 49–59. the hemolymph of Scarce Large Blues collected in Ant- 2.Bartninkaitė I. 2002. Hemolimfos struktūros parametrai vartė and Saviečiai, which could be due to a later col- prognuozuojant vabzdžių gausumą ir gyvybingumą. Žem- lection in the mentioned areas (7 days later than in dirbystė. Mokslo darbai. Vol. 3(79). P. 103–113. Joniškis). High numbers of hemocytes fulfilling the pro- 3.Bartninkaitė I. 2002. Impact of ermine moth caterpillar tective and feeding functions show that Scarce Large developing time on the dynamics of its hemolymph struc- Blue adults’ fecundity and viability will ensure the nor- ture and trophic relations in various biotopes. Ekologija. mal functioning of the population provided the feeding Nr. 2. P. 32–42. basis is suitable and the meteorological state is favor- 4.Bartninkaitė I., Ivinskis P. 2003. Pušinio pelėdgalvio (Pa- able. nolis flamea Lepidoptera, Noctuidae) lėliukių fiziologinės būklės ir masės dinamika masinio dauginimosi židiniuose. CONCLUSIONS Lietuvos biologinė įvairovė (būklė, struktūra, apsauga). P. 17–18. 1. Five types of form elements – proleucocytes, macro- 5.Bartninkaitė I., Tekoriutė B. 1994. Influence of the phy- nucleocytes, micronucleocytes, enocytoides and phago- siological state of nettle moth on its trophic relations. cytes – have been identified in the hemolymph of ca- Acta Entomologica Lituanica. Vol. 12. P. 80–88. terpillars, and only three types – macronucleocytes, mic- 6.Bauer E., Trenczek T, Dorn S. 1998. Instar-dependent ha- ronucleocytes and phagocytes – in the hemolymph of emocytes changes in Pieris brasicae after parasitization adult Scarce Large Blues. by Cotesia glomerata. Entomologia Experimentalis Appli- 2. Different percentage ratio of different type hemo- cata. Vol. 88. P. 49–58. cytes in the hemolymph of the Scarce Large Blue de- 7.Jonaitis V., Ivinskis P., Pakalniškis S., Bartninkaitė I., Pup- pends upon the stage of development. In the course of lesienė J. 2000. The interecosystematic relationships of development, the numbers of proleucocytes in the he- insects and their dynamics. Vilnius. P. 317–336. molymph decreases from 15.3 to 10.4% on average, 8.Russo J., Brchelin M., Carton Y. 2001. Hemocyte chan- and they entirely disappear in the hemolymph of adult ges in resistant and susceptible strains of the parasitic insects. The amount of macronucleocytes decreases from Leptopilina boulardi. Journal of Insects Physiology. 40.7–38.4% in the hemolymph of different size caterpil- Vol. 47. N. 2. P. 167–172. lars to 2.5–3.7% in the hemolymph of adults. The num- 9.Yamashita M., Iwabuchi K. 2001. Bombyx mori prohe- ber of micronucleocytes increases from 24–32 in the mocyte division and differentiation in individual micro- hemolymph of caterpillars to 30–37 in the hemolymph cultures. Journal of Insects Physiology. Vol. 47(4–5). of adults, and the amount of phagocytes increases from P. 325–331. 7–10 to 38–47, respectively. 10. Лакин Г. Ф. 1980. Биометрия. Москва. 3. Proleucocytes and macronucleocytes have been 11. Мисялюнене И. С., Мураускайте Г. П., 1981. Морфо- found to divide by mitosis in the hemolymph of cater- логия и функциональная активность клеток гемолимфы pillars; as caterpillars grow, mitosis is slowing down кольчатого шелкопряда в разныx фазаx постэмбрио- from 21.6 to 13.6% for proleucocytes and from 8.9 to нального развития. Тр. АН Лит. ССР. Сер. В, Т. 1(73). 5.2% for macronucleocytes. C. 33–41. 4. Based on Scarce Large Blue hemograms we can 12. Приставко В. П. 1979. Принципы и методы экспери- conclude that the physiological state of both caterpillars ментальной энтомологии. Минск. and adults in different areas of the country is good and 13. Сагды Ч. Т. 1991. Сравнительная и функциональная we expect a good fecundity and viability of the Scarce морфология гемоцитов жуков семейства чернотелки. Large Blue in this population. Кизыл. 14. Солодовникова В. С., Белоконь А. С., Маркина Т. Ю. ACKNOWLEDGEMENT и др. 1990. Динамика популяций насекомых как пока- затель окружающей среды. Вест. Харьк. ун-та. № 346. This study was financed by the Lithuanian State Scien- C. 67–69. ce and Studies Foundation (Contract No T-86/05). 15. Чхоидзе М. С. 1991. Значение гематологического мето- да в диагностике заболеваний и прогнозирования чис- Received 15 June 2006 ленности вредителей леса. Всес. науч.-техн. конф. «Ох- Accepted 20 October 2006 рана лес. экосистем и рац. использ. лес. ресурсов». Тез. докл. Москва. Ч. I. C. 126–128. Scarce Large Blue (Maculinea teleius) hemolymph studies in different populations of Lithuania 39

Povilas Ivinskis, Ilona Bartninkaitė, Jolanta Rimšaitė vystantis kraujalakiniam melsviui. Melsvio hemolimfoje proleu- kocitai buvo aptikti tik vikšrų stadijoje, o suaugėlių hemolim- KRAUJALAKINIO MELSVIO (MACULINEA TELEIUS) foje jų nebuvo. Makronukleocitų kiekis melsvio hemolimfoje HEMOLIMFOS TYRIMAS SKIRTINGOSE LIETUVOS jam vystantis taip pat mažėja. Pagal melsvio suaugėlių, surinktų POPULIACIJOSE įvairiose vietose, hemogramas, visose tirtose vietose jo fiziolo- ginė būklė yra gera, o skirtingų tipų hemocitų procentinis san- Santrauka tykis kinta nežymiai. Atlikti kraujalakinio melsvio hemolimfos tyrimai skirtingose Raktažodžiai: hemolimfa, Maculinea teleius, hemogramos, Lietuvos modelinėse populiacijose. Atlikta lervų, lėliukių ir su- hemocitai augėlių hemolimfos analizė, kuri atskleidė hemolimfos pokyčius