ACTA UNIVERSITATIS AGRICULTURAE ET SILVICULTURAE MENDELIANAE BRUNENSIS Volume LIX 9 Number 6,2011 NOTES ON DISTRIBUTION OF BOMBUS CRYPTARUM (H YMENOPTERA, APOIDEA) IN MORAVIAN TERRITORY (CZECH REPUBLIC) AND ITS LABORATORY REARING A. Bučánková, O. Komzáková, T. Cholastová, V. Ptáček Recieved: April 5,2011 Abstract BUČÁNKOVÁ, A., KOMZÁKOVÁ, O., CHOLASTOVÁ, T, PTÁČEK, V.: Notes on distribution of Bombus cryptarum (Hymenoptera, Apoidea) in Moravian territory (Czech Republic) and its laboratory rearing. Acta univ. agric. et silvic. Mendel. Brun., 2011, LIX, No. 6, pp. 69-74 B. cryptarum and B. magnus are among the so-called cryptic species whose identification is very difficult due to high interspecific variability of their morphological characteristics. This greatly limits possibilities for studying their biology, bionomics and ecology. The aim of this research was to contribute to knowledge about distribution of B. cryptarum in Moravia (Czech Republic) and to verify the possibility of its laboratory rearing. During 2006-2009, 26 collections were performed at Moravian localities. Of the 17 specimens that were assigned to B. cryptarum or B. aff. cryptarum based on morphology, the molecular analysis of mtDNA RFLP confirmed the identification of 10 speciemens. The molecular analysis even assigned to B. cryptarum one specimen determined morphologically as B. aff. magnus. Of four queens captured in spring, and while applying a laboratory method used for breeding of B. terrestris, two complete nests were successfully reared, including queens of the 2nd generation that then set up their own brood. Species identification of these queens as B. cryptarum was confirmed by both molecular analysis and analysis of the marking pheromones of males among their offspring. A single B. terrestris worker was able to raise young queens of B. cryptarum of the brood. Czech Republic, Moravian territory, faunistics, rearing, bumblebees, cryptic species, Bombus cryptarum, DNA analyses The bumblebee Bombus cryptarum belongs to the subgenus Bombus s. str. in which there exist so-called cryptic species. Generic identification of these species is often very difficult due to the large interspecific variability of their morphological characteristics. In the Czech Republic, there are three other species of this subgenus: B. terrestris (Linnaeus, 1758), B. lucorum (Linnaeus, 1761) and B. magnus (Vogt, 1911). While existence of the species B. terrestris and B. lucorum has previously been generally accepted, B. magnus and B. cryptarum were considered to be merely morphological variants of the B. lucorum species (Williams, 1991, 1998). Not until recent studies based on molecular techniques and chemical analysis of labial glands for male pheromones (Bertsch et al, 2004, 2005) has it been shown that these are indeed separate species. Generic classification of the bumblebees B. cryptarum and B. magnus on the basis of morphological characteristics, however, remains very complicated. This problem has been satisfactorily resolved only for the queens of these bumblebees (Bertsch et al, 2004, 2005). Species identification of males and workers is very difficult and not always reliable, as to date there are no fully applicable identification keys (AMIET, 1996). This issue greatly limits the possibility to obtain information about the biology, ecology and distribution of these species, which is related in turn to limited possibilities for the species' protection. 69 70 A. Bučánková, O. Komzáková, T. Cholastová, V. Ptáček From information collected in recent years, most of which is based solely on the readily identified queens, it appears that B. cryptarum is spread across Europe. This type is more common in the Benelux countries, northern and central Germany, Poland, and in some parts of Great Britain. Several records come also from Russia. It is less common in the southern parts of these countries. Information is still lacking, however, for many European countries (Bertsch et al, 2004; Rasmont et al, 1986; Rasmont, 1986,2010). Concerning knowledge of B. cryptarum's presence within the Czech Republic, the situation is similar to those in other European countries. The first faunistic information for B. cryptarum from the Czech Republic was published by Tkalců (1999). Since 2004, B. cryptarum and B. magnus began to be placed onto generic lists of Hymenoptera of the Czech Republic as two separate species (Přidal, 2004). Most existing findings of B. cryptarum come from wet overgrown meadows and pastures with bushes at altitudes ranging from 183 to 1000m a. s. 1. (Komzáková etal, 2008). Since 2006, we have been monitoring the occurrence of B. cryptarum and B. magnus in Moravia and analyzing the DNA of specimens captured while also examining the possibility of laboratory rearing for purposes of species protection as part of Ministry of Education Project No. 2B06007: Bumblebees as an active element of landscape biodiversity. MATERIALS AND METHODS Methods of capture. Bumblebee queens of the species B. cryptarum, as well as males and workers of the subgenus Bombus were collected individually by sweeping with a net at 26 selected localities in Moravia, which included submontane and lowland habitats (Tab I). The queens for laboratory rearing were collected in spring when seeking a nest. Collection of males and workers was conducted in summer. The captured specimens were identified by Antonín Přidal from Mendel University in Brno. Collected material has been stored in a dry state in entomological boxes and in this condition it was used for molecular analysis. Molecular determination of species using RFLP analysis. Genomic DNA was isolated from two legs of each specimen/individual using the commercially available DNeasy"1 Blood <& Tissue Kit (Qiagen, Germany). A part of the mitochondrial COl gene (1064 bp) was amplified by PCR using the primer pair COl.f orward 5' -ATAATTTTTTTTATAGTTATA- 3' and COl.reverse 5' - GATATTAATC C TAAAAAA TGTTGAGG-3' (Tanaka et al, 2001). Individual PCR was carried out in 30 ul volumes containing lOx buffer (including 1.5 mM MgCLj (Finnzymes, Finland), 200 uM of dNTP mix (Finnzymes, Finland), 2 uM of each primer (Metabion, Germany), 0.2 U of DyNAzyme II DNA polymerase (Finnzymes, Finland), and 2 ul of template DNA. The PCR cycle was as follows: 93 °C for 1 min, followed by 30 cycles of 93 °C for 45 s, 45 °C for 1 min and 60 °C for 4 min, with a final 3 min extension period at 60 °C. PCR products were run out on a 1.5% agarose gel and visualized over UV light after staining in ethidium bromide. Amplified mtDNA fragments were digested using the restriction enzyme Hinf I. Each reaction contained 7.5 ul of PCR product, 1U of Hinf I enzyme and 1 ul of lOx NEBuffer 2 (New England Biolabs Inc.). HPLC-grade water was added to obtain a total reaction volume of 10 ul. The samples were incubated at 37 °C for 4 h. Restriction fragments were electrophoretically separated on 2% agarose gel and visualized over UV light after staining in ethidium bromide. For comparison, DNA were used that were obtained from one B. cryptarum individual whose identity had been verified by the male marking pheromone, one B. terrestris individual from laboratory rearing at Masaryk University, and one B. lucorum individual captured in nature and identified based on morphological characters. Determination using pheromones (Urbanova etal, 2001; Terzo etal, 2005; Rasmont etal, 2005) was possible in males from laboratory rearing and was carried out by the Institute of Organic Chemistry and Biochemistry (IOCB) of the Academy of Sciences of the Czech Republic in Prague. Rearing methodology was the same as for commercially bred species of B. terrestris. Captured queens were placed into a plastic rearing box with dimensions of 140 x 190 x 70 mm and kept in darkness at 27-28 °C and 60-80% relative humidity They were fed 60% sugar solution (90% sucrose, 10% fructose) and fresh pollen, which was replaced daily Only in the case of one wintering queen was dried and frozen pollen used. To stimulate laying, they were offered a cocoon of B. terrestris, or possibly B. lucorum. In 2007, after laying eggs one queen received as a helper a callow worker of B. terrestris. Once the first workers had hatched, the colonies were moved into wooden hives with dimensions 265 x 200 x 200 mm. Young queens emerging in the hives were moved along with unrelated males into a glass aquarium covered by a netting in daylight to mate. Queens whose mating was observed were removed from the terrarium and transferred to rearing boxes in darkness at room temperature. Here, they were provided with sugar solution in order to create energy reserves for the period of hibernation. Queens who had been observed to mate were left with males for approximately 10 days, after which they were moved to the same conditions as the queens who had mated. Queens showed readiness to enter hibernation (i.e. filled abdomen and minimal physical activity) were after 7 days placed into a refrigerator at 4 °C. The queens, who remained remarkably active, were thereby stimulated to lay eggs as were the queens caught in the wild. Notes on distribution of Bombus cryptarum (Hymenoptera, Apoidea) in Moravian territory (Czech Republic) 71 RESULTS AND DISCUSSION During 2006-2009 as part of the project, we managed to capture a total of 17 bumblebee specimens (3 workers, 14 males). Based on morphological characteristics, they were assigned to B. cryptarum or species of B. aff. cryptarum. Four of the specimens (1 worker, 3 males) were identified as B. magnus or B. aff. magnus. In 11 cases, restriction digestion of a part of the mtDNA COl gene with the enzyme Hinf I resulted in two restriction fragments which differed in length, one being 500 bp and the other 550 bp. The presence of these two bands confirmed the identity of B. cryptarum. Two individuals were assigned to B. terrestris. In 3 individuals identified on the basis of morphology as B. magnus and 5 specimens identified as B. cryptarum or B. aff. cryptarum digestion did not occur, and the result was the undigested PCR product 1064 bp in length. This suggests that these most likely are not individuals of B. cryptarum, because in the given sequence restriction sites characteristic of these species were not found. But whether or not these are B. magnus remains to be verified. A reliable method for determining the species' generic identity is by chemical analysis of the labial glands for male pheromones, but this requires sampling the head tissue immediately after death, which in this case was possible only for specimens obtained from laboratory rearing. I: Localities, date of collection and comparison of morphological identification and mtDNA analysis Locality Date of collection Morphological identification PCR-RFLP NP Podyjí, pod Šobesem 17. 8.2006 B. cryptarum B. terrestris NP Podyjí, Zadní Hamry, pod Led.slujemi 17. 8.2006 B. cryptarum B. cryptarum CHKO Žďárské vrchy, Světnovské údolí 2. 9.2006 B. cryptarum B. cryptarum CHKO Žďárské vrchy, Světnovské údolí 2. 9.2006 B. magnus - CHKO Žďárské vrchy, Světnovské údolí 2. 9.2006 B. magnus - CHKO Žďárské vrchy, Světnovské údolí 2. 9.2006 B. magnus - Slavice, okr. Třebíč 11. ó. 2007 B. cryptarum - CHKO Žďárské vrchy, Světnov louka 13. ó. 2007 B. cryptarum B. cryptarum CHKO Bílé Karpaty, Strání 4. 8.2007 B. cryptarum B. terrestris Obecní díly, nad Šternberkem směr Nové Dvorce 28. 4.2008 B. cryptarum B. cryptarum Obecní díly, nad Šternberkem směr Nové Dvorce 28. 4.2008 B. cryptarum B. cryptarum CHKO Žďárské vrchy, Radostín 5. 7.2008 B. aff. cryptarum B. cryptarum NP Podyjí, Mašovice 29. 7.2008 B. aff. cryptarum B. cryptarum Orlické hory, Šerlich- Orlické záhoří 16. 8.2008 B. aff. cryptarum - CHKO Žďárské vrchy, Radostín 31. 8.2008 B. aff. cryptarum B. cryptarum Obecní díly, nad Šternberkem směr Nové Dvorce 12. 8.2009 B. aff. cryptarum - Obecní díly, nad Šternberkem směr Nové Dvorce 12. 8.2009 B. aff. cryptarum - CHKO Jeseníky, Karlov 12. 8.2009 B. aff. cryptarum - CHKO Žďárské vrchy, Stržanov 14. 8.2009 B. aff. magnus B. cryptarum CHKO Žďárské vrchy, Světnovské údolí 14. 8.2009 B. aff. cryptarum B. cryptarum CHKO Žďárské vrchy, Světnovské údolí 14. 8.2009 B. aff. cryptarum B. cryptarum NP = national park, CHKO = protected landscape area The presence of B. cryptarum was found across Moravia at altitudes between 200 and 1000 m. a. s. 1., but they are rather rare (see Table I.). As stated in the methodology section, four queens were available to verify the laboratory rearing of B. cryptarum. Two queens were captured in the spring of 2007 (one at the site Zálesí, between Moravský Beroun and Krahulci, and one at the site Obecní díly, nad Šternberkem směr Nové Dvorce (Municipal lands outside of Šternberk in the direction of Dvorce). At the second of these sites, two queens were found in spring 2008. Queens found in 2007 were determined only morphologically and are not listed in the table. In 2007, one of the queens died after 8 days in the laboratory, probably due to ill health. The second queen established brood and after 7 days she was given a callow worker of B. terrestris to help her with care of the brood. Adoption of the B. terrestris worker by the B. cryptarum queen and subsequent cooperation in the care of the brood were apparently successful. After establishing the egg cell, however, the queen died and the worker raised five young queens of the orphaned brood. Since at that time B. cryptarum males were not available, queens were offered B. lucorum and B. terrestris males to mate. Although de Jongh (1982) and Rasmont <& de Jongh (1985) had reported queens mating with B. terrestris and B. lucorum males, we recorded no interspecific mating. Two of these five young queens of the 2nd 72 A. Bucdnkovd, O. Komzdkovd, T. Cholastovd, V.Ptdceh generation in the autumn showed a behavior that usually leads to eggs laying but they laid no eggs and eventually died. In contrast, both the queens obtained in 2008 began unhesitatingly to establish egg cells on the B. terrestris cocoons. They raised their own workers and after transfer to the hive were developed well. After producing several generations of workers, the families began to produce variably colored males and later young queens as well. The colonies' time courses of development, numbers of queens, and numbers of individuals in the nests are shown in Table II. At the time of producing young queens, there were young males in both colonies, so it was possible to attempt their mating under laboratory conditions. Mating was observed in only 6 of the total number of 32 queens. One queen died soon after mating. In addition, queens after mating did not show typical behavior for queens of B. terrestris, which generally are ready to enter hibernation. Seven queens were still wintering in the refrigerator and the others were placed in the breeding room, where seven started to lay eggs. Three queens which had produced brood nevertheless died after a few days, two raised only the first generation of workers, and the remaining two raised only males. Of the seven queens that were wintering in the laboratory in early September, just one was still living at the beginning of the following February. She did not establish the egg cells, however, and died a few days after coming out of hibernation. Chemical analysis performed at IOCB of the male labial glands' pheromones from both colonies (Table II) and from males reared by queens of the second generation confirmed that they were B. cryptarum. II: Time behavior and number of individuals in colonies originating from B. cryptarum queens collected in 2008 Colony Date of collection 1st egg cell 1st worker 1st queen Number of queens produced In colony Total number of Individuals on date 26.8.08 A 28.4.08 7.5. 28.5. 14.7. 12 290 B 28.4.08 6.5. 28.5. 27.6. 10 160 SUMMARY The species B. cryptarum and B. magnus are among the so-called cryptic species whose identification is very difficult due to their high interspecific morphological variability. During 2006-2009, the occurrence in Moravia of the cryptic species B. magnus and B. cryptarum was observed. Bumblebees were collected at 26 habitats of submontane and lowland types. Collection was conducted in spring, when the queens could be obtained for laboratory rearing, and in summer, when male and worker were obtained for determination. Males and workers were determined based on morphological characteristics. Consequently, their generic affiliation was verified by mtDNA RFLP analysis at the Research Institute for Fodder Crops, Ltd. and Agricultural Research, Ltd. in Troubsko, Czech Republic. The young queens collected were used for rearing at the Faculty of Science, Masaryk University, in Brno. By means of morphological determinations, 17 specimens were assigned to the species B. cryptarum or o/B. aff. cryptarum and 4 specimens were assigned to the species of B. aff. magnus. Molecular analysis of a part of the mtDNA COl gene using RFLP confirmed the identity of B. cryptarum in 11 individuals while 2 individuals were identified as B. terrestris. One individual morphologically designated as B. aff. magnus was identified as B. cryptarum. In 8 individuals, digestion did not occur in a part of the mtDNA COl gene, thus indicating a high probability that this was not of the B. cryptarum species. Whether or not it is B. magnus remains to be verified. Four B. cryptarum queens were captured during 2007 and 2008 (two in each year). These were taken to the laboratory for experiments using methods practiced for B. terrestris. In 2007, one queen died. A B. terrestris worker was added to the second one, which already had its own brood. Subsequently, that queen also died and the worker raised five young queens from the brood. In 2008, both captured queens succeeded in rearing two colonies, which produced sexual specimens. Attempts at mating of the young queens were only partially successful. Moreover, the mated queens did not show typical pre-hibernation behavior common for B. terrestris. Some of the queens subsequently began to lay eggs and raised a number of workers and males. Of the seven queens wintering like B. terrestris only one survived, and after a few days that one died in the laboratory. The correct identification of these two bumblebee colonies as B. cryptarumwas confirmed by analyzing the marking pheromone from males of the second generation. Notes on distribution of Bombus cryptarum (Hymenoptera, Apoidea) in Moravian territory (Czech Republic) 73 Acknowledgments Many thanks to Ing. Klára Urbanová, PhD. (Institute of Organic Chemistry and Biochemistry, Academy of Sciences) for carrying out analyses of male marking pheromone. The findings are the outcome of research project No. 2B06007 and No. MSM2629608001 funded by the Ministry of Education, Youth and Sports of the Czech Republic. REFERENCES AMIET, E, 1996: Hymenoptera, Apidae. 1. Allgemeiner Teil, Gattungsschlüssel, die Gattungen Apis, Bombus und Psithyrus. Fauna Helvetica 3. 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Olga Komzáková, Výzkumný ústav pícninářský, spol. s r. o. Zahradní 1, 66441, Troubsko, Česká republika, Mgr. Alena Bučánková, Mgr. Tereza Cholastová, Zemědělský výzkum, spol. s r. o., Zahradní 1, 664 41, Troubsko, Česká republika, doc. RNDr. Vladimír Ptáček, ÚEB, Přírodovědecká fakulta, Masarykova univerzita, Kotlářská 2, 611 37 Brno. Zemědělský výzkum, spol. s r. o., Zahradní 1, 66441, Troubsko, Česká republika, e-mail: komzakova@vupt.cz, bucankova@vupt.cz, cholastova@vupt.cz, ptacek@sci.muni.cz 74