Studying on the component of honey bee species (hymenoptera: apidae) and the ability to use some species as an indicator estimating environmental pollution on affected ecosystems in the north of vietnam

ratina 4 chiangmaiensis was discovered and identification key for each genus were also provided. According to the list of honey bee species counted up by two scientists Ascher and Pickring, genus Ceratina currently has 366 species distributing all over the world; genus Amegilla includes 247 species belonging to 12 sub-genera, distributing all over the world (according to Ascher and Pickering, 2016). 1.2.1.2. Studies on the possibility for using honey bee species as biological indicators of environment Studies of Conti and Botre (2001) on using products of Apis mellifera (honey, pollen and beeswax) in Roma (Italy) were conducted through identifying contents of heavy metals such as Cd, Cr and Pd in order to estimate environmental pollution in this city. Another study on using bodies of Apis mellifera to watch over environmental pollution in Italy determined 250 individuals of honey bees died per week at an investigated site because of using pollen and nectar from pesticide polluted plants (Porrini et al., 2003). Furthermore, there were some research results on this topic of Ghini et al. (2004) , Madras (2005), Chauzat et al. (2006), Chauzat and Faucon (2007), Popescu et al. (2010), Zhelyazkova (2012), Ruschioni et al., (2013), Naccari et al., (2014) and Moniruzzaman et al., (2014). 1.2.1.3. Studies on ecological relationship and conservation of Apidae honey bee species Studies on this domain include publications of Vivian and Butz (1997), Parker et al., 2010), Becher et al., (2010), Radloff, 2011), Radloff, (2011), Shaara (2014), Loren et al. (2016) and Juliano Nogueira (2014). Worker bees occupied a large proportion in bee colonies should be the main objects in the analysis and study of morphology and biological norms of honey bee (Ruttner, 1988). Feculity of queen bees is a very important indicator to estimate developing capacity of bee colonies as well as the quality of bee breeds (according to Shaara, 2014). 1.2.2. The studies in Vietnam 1.2.2.1. Studies on species component and distribution of Apidae honey bee species + Studies on species component In Vietnam, studies on species component and distribution of Apidae honey bee species have not been paid much attention. Up to now, there have been only 3 studies on the fauna of Apidae honey bee species in Vietnam. Le Xuan Hue (2008) counted up 41 species belonging to 11 genera of Apidae which were recorded, among them 25 species belonging to 8 genera recorded in Northern Vietnam. In 2010, one species of genus Bombus was described as a new species for science which based on the specimen of Vietnam (Le Xuan Hue, 2010). Recently, 35 species belonging to 9 genera have been recorded for the fauna of Apidae honey bee species in the North and Central of Vietnam (Khuat Dang Long et al., 2012), among them 32 species belonging to 9 genera have been counted up in Northern. 5 + Studying on honey bees bred to get honey and their distribution In Vietnam, for many years studies on honey bees have focused on species of honey bees breeding for nectar. Phung Huu Chinh and Pham Thi Huyen (2004) divided breeding honey bees species into: dwarf honey bee with 2 species (red dwarf honey bee Apis florea, black dwarf honey bee Apis andreniformis), giant honey bee Apis dorsata, Himalayan giant honey bee Apis laboriosa, domestic honey bee Apis cerana, exotic honey bee Apis mellifera. In Vietnam there are 4 main species of honey bee including European honey bee (exotic honey bee) Apis mellifera, domestic honey bee (Asiatic honey bee) Apis cerana, giant honey bee Apis dorsata and drwarf honey bee Apis florea. According to Nguyen Van Niem (2014), in Vietnam, domestic honey bee Apis cerana includes two sub-species: A. cerana indica Fabricius, 1798 and A. cerana cerana Fabricius, 1793. Some studies of Pham Hong Thai (2008) showed that in Ly Son island (Quang Ngai), Phu Quy island (Binh Thuan) and Ca Mau peninsula domestic honey bees have not been discovered. 1.2.2.2. Studies on the possibility for using honey bee species as biological indicators of environment In Vietnam, the possibility for using honey bees as biological indicator in order to estimate environmental pollution has not been paid much attention and has been poorly studied. Up to now, there have not been any research results on contents of heavy metals and metalloid inside bee bodies, guts, beeswax and honey. The beginning studies were at standstill in the study to determine the chemical pesticides belonging to Clo group existing in longan flower honey in Hung Yen (Phung Huu Chinh and Dinh Quyet Tam, 2004, 2007) and determining polluted limits of some metals and metalloids existing in honey, including Cd, Pb, Hg and As (Vietnam Food Administration, 2011) 1.2.2.3. Studies on ecological relationship and conservation of Apidae honey bee species Studies on ecological relationships and conservation of Apidae honey bee species only concentrated on honey bee species breeding for nectar such as: domestic honey bees Apis cerana, Exotic honey bees Apis mellifera, red dwarf honey bee Apis florea, giant honey bee Apis dorsata (Ha Thi Hue et al., 2010). Honey bees are social insects, bee colonies is an integrated biological unit with three closely connected kinds of bees including queen bee, male bees and worker bees (Crane E., 1990; Le Quang Trung and Nguyen Tuong Van, 2012). According to Nguyen Duy Hoan, 2002 and Nguyen Duy Hoan et al., 2008 provided data on ecological forms under the influence of different surrounding factors and characteristics adapting to different living conditions of sub-species of domestic honey bees A.cerana and exotic honey bee A.mellifera. Studies on structures of bee colonies, biological characteristics, techniques to catch forest bees in order to breed, techniques creating queen bee, dividing colonies of domestic species of honey bees Apis cerana, exotic Apis mellifera, red dwarf 6 honey bees Apis florea, giant honey bees Apis dorsata were also carried out (Phung Huu Chinh and Pham Thi Huyen, 2004). 1.2.2.4. Some remarks on studying Apidae honey bee species Up to now, in Vietnam 41 species of honey bees belonging to the family Apidae, 11 genera among them 35 species belonging to 9 genera have been recorded in the North and Central of Vietnam. Other studies only concentrated on species of honey bee breeding for nectar such as: domestic honey bees Apis cerana, exotic honey bee Apis mellifera, red dwarf honey bee Apis florea, giant honey bee Apis dorsata. Research results on the possiblity for using honey bee as biological indicator in order to estimate environmental pollution has not been paid much attention and has been poorly studied. Chapter 2 LOCATION, TIME AND METHODS OF STUDYING 2.1. Location and time of studying Carrying on investigation to collect specimens in some affected ecosystems in 18 provinces in Northern Vietnam such as: Hanoi (The middle field of the Red river, Long Bien, Truc Khe, Lang Ha, Ba Vi National Park, Hoai Duc), Phu Tho (Dong Thinh, Yen Lap), Bac Ninh (Tu Son, Tien Du, Que Vo), Hoa Binh (Yen Thuy, Mai Chau, Da Bac), Yen Bai (Yen Binh, Tan Phuong, Viet Hong), Son La (Moc Chau, Muong Lum, Yen Chau, Co Ma, Thuan Chau), Lai Chau (Muong Te, Sin Ho), Dien Bien (Tua Chua, Quang Lam), Lao Cai (near Thac Bac, Hoang Lien Son National Park, Sapa, Lao Cai), Vinh Phuc (Tam Dao National Park), Ha Giang (Quan Ba, Dong Van), Cao Bang (Phia Oac, Nguyen Binh) Tuyen Quang (Nui Dum, Na Hang), Bac Kan (Lang Ngam, Ngan Son, Kim Hy, Na Ri), Thai Nguyen (Phu Luong), Lang Son (Mau Son, Huu Lung), Bac Giang (Khe Ro, Son Dong), Quang Ninh (Dong Trieu, Ba che, Tien Yen). Experimental stidies, data processing have been carried out in the Department of Entomological Ecology, Institute of Ecology and Biological Resources and the Laboratory of Chemistry Analyzing, Institute of Military Environmental Chemistry. The thesis has been studied from 2014 to 2017. 2.2. Materials of studying Tools and equipments for investigation and experiments on the laboratory include: Insect nets, glass tubes, glass tube holder, big trays to contain specimens, petri and triangle, abrasive pack bottles, plastic vials to contain specimens, killing insect vials (with toxin killing insects Ethyl acetate, Naphthalene), cotton pads to keep specimens, vials to preserve specimens, pins, scissors, brushes, dissecting needles, different sized net cages, microscope Olympus 52x7, two eyed magnifier, automatic thermo-hygrometer incubator Sanyo and Wise cuber, heating apparatus, lamps and refrigerator, cotton netting, rice paper, absorbed cotton and disabsorbed cotton, notebook to record investigated and experimental data. 7 2.3. Methods of studying 2.3.1. Methods of investigating species component, distribution and describing morphological characteristics of new recorded species 2.3.1.1. Investigating and collecting specimens in the field We used the common methods of investigating insects, including : method of investigating and discovering insects, process and techniques to collect, process and preserve insects, basically methods of investigating insects, methods of Grootaert et al.(2010) including: lines survey, habitats survey and collecting methods. 2.3.1.2. Preservation, making insect sample and classification + Preservation and making insect samples: Preservation and making insect sample with cotton pads, 70° alcohol, 90° alcohol for DNA studies. + Morphological classification: Based on the documents of Michener (2005, 2007), He (2004). + Molecular biological classification: DNA samples after being purred and amplified, were sent to read at 1st BASE company (Malaysia). The DNA sequence were analyzed and processed with Bioedit software. 2.3.1.3. Describing some morphological characteristics of new recorded species Based on the morphological characteristics of head, thorax, scutellum, abdomen and wings. 2.3.2. Methods of studying the possibility for using some honey bee species as biological indicator Selecting indicator species according to Chauzat et al. (2006) and Ghini et al. (2004) 2.3.2.1. Methods of community investigating and estimating potential of using Interviewing directly local people for gathering information. 2.3.2.2. Examining contents of heavy metal on the bee’s bodies and honey products Inductively Coupled Plasma Emission Mass Spectrometry (ICP-MS) (EPA, 2007), Atomic absorption spectrophotometry, Analytical methods ICP-MS (TCVN 7602:2007, TCVN 7603:2007, TCVN 7604:2007; high performance liquid chromatography (HPLC), gas chromatography mass spectrometry GCMS 2.3.3. Methods of studying ecological relationship of honey bees species with different habitats Groups of habitats for studying ecological relationship include: (1) Evergreen forest associated with the ecosystem of natural forest (SC1) (2) Cultivated forest associated with the ecosystem of cultivated forest (SC2) (3) Perennial garden associated with the ecosystem of perennial garden (SC3) (4) Annual crops (rice, corn, bean, peanut, sugarcane, jute, vegetables...) associated with the ecosystem of the field (SC4) Using methods to collect and catch insects and analyze data as in section 2.3.1 2.3.4. Methods of processing data and calculating formulas The collected data were processed, analyzed and calculated on the computer according to mathematical methods applied in biology with the level of probability 8 P<0.05 and some biological statistics software such as: SPSS 9.0 and RESTART 4.0 in Windows 2000. Chapter 3 RESEARCH RESULTS AND DISCUSSION 3.1. Species component and distribution of Apidae honey bee species in some provinces in Northern Vietnam 3.1.1. Species component and distribution of Apidae honey bee species at investigated sites In habitats surveyed in 18 province of Northern Vietnam, 60 species of honey bees belonging to 2 sub-families (Apinae and Xylocopinae) and 7 genera belonging to sub-family Apidae were recorded. 10 species were new recorded for the fauna of honey bee in Vietnam, including: Thyreus abdominalis rotratus, Thyreus ceylonicus lilanius, Thyreus decorus, Thyreus centrimacula, Thyreus regalis, Elaphropoda khasiana, Ceratina lieftinck, Ceratina collusor, Ceratina humilor, Ceratina sutepensis. In the North, together with above mentioned 10 species research results recorded more 7 new species for the fauna of honey bee, including: Amegilla himalajensis, Ceratina smaragdula, Ceratina simillima, Ceratina unimaculata, Xylocopa aestuans, Xylocopa dejeanii, Xylocopa ruficornis). Especially, the species Thyreus regalis was recorded in Me Linh, Vinh Phuc province and this was an important information suplementing data on the distribution of this species on the distribution world map. The structure of species component following genera includes: genus Ceratina with the most number of species - 17 species, genus Xylocopa with 11 species, genus Thyreus with 9 species, genus Bombus with 7 species, genus Amegilla with 6 species, genus Apis with 5 species and genus Elaphropoda with 5 species. However, the highest catch rate (%) belonged to genus Apis (35,12%) and the lowest catch rate belonged to genus Elaphropoda (0,04%) (Figure 3.1). Figure 3.1. Structure of species component of Apidae in the North Vietnam 9 Figure 3.2. The catch rate (%) of Apidae honey bee species in Northern provinces of Vietnam Investigating of the distribution and catch rate of honey bee species in 20 Northern provinces showed that: Apis cerana had the largest distribution and appeared in 16/20 investigated provinces, the next were exotic honey bee Apis mellifera, giant honey bee Apis dorsata and bumblebee Bombus sp. distributed in 8/20 investigated provinces, dwarf honey bee Apis florea distributed in 7/20 investigated provinces. The rest species distributed in 1-3 investigated provinces. (Figure 3.2) 3.1.2. Morphological characteristics of new recorded species in Vietnam 3.1.2.1. Morphological characteristics of Ceratina collusor Cockerell, 1919 Female Figure 3.3. Morphological characteristics of Ceratina collusor A. Head E. Prothorax B. Coxa of foreleg F. Metanotum C. Space between simple and compound eye G. Abdomen, dorsal view D. Thorax, dorsal view H. Body 3.1.2.2. Morphological characteristics of Ceratina humilor Cockerell, 1916 Female 10 Figure 3.4. Morphological characteristics of Ceratina humilor A. Head, front view E. Metanotum B. Labrum F. Abdomen, dorsal view C. Prothorax G. Body D. Head and thorax, dorsal view 3.1.2.3. Morphological characteristics of Ceratina sutepensis Cockerell, 1929 Female Figure 3.5. Morphological characteristics of Ceratina sutepensis A. Head, front view D. Space between simple and compound eye B. Head, lateral view E. Abdomen, dorsal view C. Abdomen, dorsal view F. Body 3.1.2.4. Morphological characteristics of Ceratina lieftinck van der Vecht, 1952 Female Figure 3.6. Morphological characteristics of Ceratina lieftinck 11 A. Head, front view D. Space between simple and compound eye B. Head, lateral view F. Body C. Abdomen, dorsal view 3.1.2.5. Morphological characteristics of Elaphropoda khasiana (Schulz, 1906) Female Figure 3.7. Morphological characteristics of Elaphropoda khasiana A. Head, lateral view D. Trochanter of hindleg B. Antenna E. Abdomen, dorsal view C. Wing F. Body 3.1.2.6. Morphological characteristics of Thyreus abdominalis rostratus (Friese, 1905) Male Figure 3.8. Morphological characteristics of Thyreus abdominalis rostratus A. Head, front view D. Tibia of middle leg B. Metanotum E. Abdomen, dorsal view C. Spiracle IV-VII, dorsal view F. Body 3.1.2.7. Morphological characteristics of Thyreus ceylonicus lilacinus (Cockrell, 1919) Female 12 Figure 3.9. Morphological characteristics of Thyreus ceylonicus lilacinus A. Head, front view (male) E. Thorax, dorsal view (female) B. Thorax, dorsal view (male) F. Abdomen, lateral view (female) C. Abdomen, dorsal view (male) G. Male’s body D. Head, front view (female) H. Female’s body 3.1.2.8. Morphological characteristics of Thyreus centrimacula (Pérez, 1905) Female Figure 3.10. Morphological characteristics of Thyreus centrimacula A. Head, front view C. Body B. Tibia of middle leg and hindleg 3.1.2.9. Morphological characteristics of Thyreus decorus (Smith, 1852) Female 13 Figure 3.11. Morphological characteristics of Thyreus decorus A. Head, front view C. Tibia of hindleg, external view A. Abdomen, dorsal view D. Body 3.1.2.10. Morphological characteristics of Thyreus regalis Lieftinck, 1962 Female Figure 3.12. Morphological characteristics of Thyreus regalis A. Thorax, front view D. Spiracle VI, underneath view B. Thorax, dorsal view E. Body C. Metanotum 3.1.3. Molecular biological characteristics of population of domestic honey bee Apis cerana in Northern provinces, Vietnam To evaluate the diversity of bee population studied, PCR was used to amplify the segment of cox1 mitochondrial gene with COI primers. The results showed that a specific band was the right size (Figure 3.13) 14 Figure 3.13. PCR product of Cox1 gene on agarose gel 1% Analysis of nucleotide sequences by using BioEdit ver. 7.0.9 software (Figure 3.14). (a) Sequences of cox1 mitochondrial gene of honey bee sample in Ba Vi (b) Sequences of cox1 mitochondrial gene of honey bee sample in Lang Son (c) Sequences of cox1 mitochondrial gene of honey bee sample in Vinh Phuc (d) Sequences of cox1 mitochondrial gene of honey bee sample in Dong Van (f) Sequences of cox1 mitochondrial gene of honey bee sample in Cao Bang Figure 3.14. Sequences of cox1 mitochondrial gene of 5 domestic honey bee samples Apis cerana The relationship between populations of domestic honey bee species was determined basing on the sequences of cox1 mitochondrial gene, the phylogenetic tree is shown in Figure 3.15: 5 populations of studied domestic honey bee make up 2 different genetic groups. Group 1: Ba Vi, Cao Bang, Vinh Phuc; Group 2: Dong Van - HaGiang, LangSon. 15 Figure 3.15. The alignment 5 results of honey bee populations studied (The colored nucleotide posiyions are the mutated spots) BaVi CaoBang VinhPhuc DongVan-HaGiang LangSon Figure 3.16. Grouping of 5 populations of domestic honey bee in some Northern provinces according to the sequence cox1 mitochondrial gene 3.2. Studies on the possibility for using honey bee species as biological indicators to estimate environmental pollution in studied habitats 3.2.1. Honey bee species can be used as biological indicators in studied sites A total of 9 Apidae honey bee species which could be used as biological indicator to determine heavy metal contents and pesticide residues to assess environmental pollution for studied habitats (Table 3.3). Table 3.3. Honey bee species can be used as biological indicators in studied sites in the North of Vietnam N° Latin names Distribution Catch rate (%) Studied habitats SC1 SC2 SC3 SC4 1. Apis cerana Fabricius HG, CB, TQ, LS, VP, BK, QN, HN, TN, PT, SL, LCh, LC, TH, NA, VP 17,97 + + + + 2. Apis mellifera Linnaeus LS, HN, ĐB, SL, CB, 6,07 + + + + 16 3.2.2. Study on heavy metal contents in Apis cerena indica products at some studied sites Study on heavy metal contents in Apis cerena products (sub-species Apis cerana indica) was conducted in studied sites at 3 provinces Hanoi, Cao Bang and Ha Giang. 3.2.2.1. Heavy metal contents in body of Apis cerana indica In Hanoi, the order of 10 heavy metals studied in body of Apis cerana was: Fe >Zn >Mn > Cu >Hg>Ni >Pb>As> Co> Cd. The results showed that, in Hanoi 3 heavy metals Fe, Zn and Mn was high and fluctuate in content, the other elements were low in content with small amplitude (Figure 3.17). In Cao Bang, the order of 10 heavy metals studied in body of Apis cerana indica was: Fe >Zn >Mn >Cu >Pb>Ni >Co>As >Hg >Cd. Three heavy metals 3 Fe, Zn, Mn were low in content with small amplitude, Fe was found in honey bee body with high in content that determined at Tam Kim in 2015. As and Cd were found with low in content at Minh Thanh. Figure 3.17. Fluctuation of heavy metal content in body of Apis cerana indica at studied sites BK, HG 3. Apis florea Fabricius LS, HN, ĐB, SL, CB, BK, HG 6,07 + + + + 4. Apis dorsata Fabricius HG, TQ, HN, SL, VP, QN 4,76 + + + + 5. Amegilla zonata (Linnaeus) HG, CB, TQ, LS, BK, BG, BN, HN, SL 6,32 + + + + 6. Bombus flavescens Smith CB 5,58 + + 7. Thyreus himalayensis (Radoszkowski) CB, BK, BG, SL 2,13 + + 8. Ceratina nigrolateralis Cockerell LS, HB, LC, ĐB 2,71 + + 9. Xylocopa tenuiscapa Westwood CB, HN, LC, SL, PT 3,53 + + 17 In Ha Giang, the order of 10 heavy metals studied in body of Apis cerana indica was Fe >Zn >Mn >Cu >Hg >Pb >Ni >As >Cd >Co. 3 heavy metals Fe, Zn, Mn were higher in content in Cao Bang and lower in content in Hanoi (Figure 3.17) 3.2.2.2. Heavy metal content in gut of Apis cerana indica In Hanoi, the order of 10 heavy metals studied in body of Apis cerana indica was Mn >Zn >Fe >Cu >Ni >Hg >Pb >As >Cd >Co. The content of Mn, Zn, Fe were found in honey bee’s gut with big amplitude, whereas Co, As, Cd, Ni and Pb were found with smaller amplitude in content (Figure 3.18). IN Cao Bang, the order of 10 heavy metals studied in body of Apis cerana indica was Mn> Zn >Fe >Cu >Pb >Co >Hg >Ni >Cd >As. The content of 10 studied metals in honey bee’s gut in Cao Bang was found with small amplitude. Figure 3.18. Fluctuation of heavy metal content in gut of Apis cerana studied sites The research results in Cao Bang recorded 3 heavy metals Mn, Zn and Fe which were found gut of Apis cerana with high and small amplitude in content (Figure 3.18). In Ha Giang, the order of 10 heavy metals studied in body of Apis cerana indica was Mn >Zn >Fe >Cu >Hg >Cd >Pb >Ni >As >Co. The content of 10 studied heavy metals was found in gut of Apis cerana with smaller amplitude than it in Cao Bang (Figure 3.18). 3.2.2.3. Heavy metal content in beeswax In Hanoi, the order of 10 heavy metals studied in body of Apis cerana indica was Fe> Zn> Cu >Ni >Mn >Pb> Hg >As >Co >Cd. The content of Fe, Zn, Cu, Ni and Mn was found in beeswax with big amplitude. The content of Co, As, Cd and Pb was found in beeswax with smaller amplitude (Figure 3.19). In Cao Bang, the order of 10 heavy metals studied in body of Apis cerana indica was Fe >Zn >Mn >Cu >Ni >Pb >Hg >As >Co >Cd; 4 heavy metals Fe, Zn, Mn and Cu were found in beeswax of Apis cerana indica with high content and small amplitude (Figure 3.19). 18 Figure 3.19. Fluctuation of heavy metal content in beeswax of Apis cerana indica in studied sites In Ha Giang, the content of heavy metal on average was lower than Hanoi and Cao Bang. The highest content of heavy metal was Fe, then Zn and Mn. The order of 10 heavy metals studied in body of Apis cerana indica là Fe >Zn >Mn >Cu >Pb >Ni >Hg >As >Co >Cd. The content of 10 heavy metals was found in beeswax in Ha Giang with small amplitude (except FR and Zn) (Figure 3.19). 3.2.2.4. Heavy metal content in honey In Hanoi, the highest content of heavy metal on average was Fe, then Zn, Hg, Cu and Mn. The order of 10 heavy metals studied in body of Apis cerana indica là Fe >Zn > Cu >Ni> Mn> Pb >As >Hg>Co >Cd. The content of Fe was found in honey with big amplitude, whereas Hg, Co, As, Cd and Pb were found in honey with small amplitude (Figure 3.20). In Cao Bang, the content of heavy metals on average was lower than in Hanoi, the highest was Fe, then Hg, Mn and Zn. The order of 10 heavy metals studied in body of Apis cerana indica là Fe> Mn> Zn> Cu> Ni> Pb> As>Hg> Co> Cd. The content of 10 studied heavy metals was found in honey with small amplitude (Figure 3.20). Figure 3.20. Fluctuation of heavy metal content in honey of Apis cerana indica in studied sites 19 In Ha Giang, the highest content of heavy metal on average was Fe then Zn and Hg. The order of 10 heavy metals studied in body of Apis cerana indica là Fe >Zn >Cu >Mn >Pb> Ni>As > Hg >Co >Cd. The content of 10 studied heavy meta