Progress in the study of ecology, zoogeography, group, control repellent and medical usage of Hirudinea in China

Hirudinea in China were reviewed. The ecological distribution of Hirudinea, the population dynamics of H. hainana, the adaptation of H. hainana to temperature, soil humidity and pH, the response of H. hainana to temperature, relative humidity and light, and the effects of human activities on H. hainana populations were discussed. The land-leeches' behavior ecology, including (1) the locomotion of H. hainana such as shortening, oscillating, rotating, sucking cleaning, rubbing and foraging behaviors; (2) the factors influencing these behaviors; (3) the ecological significance, was also discussed. The ecology of freshwater leeches including (1) behavior in paddy fields of Ninghsien, Zhejiang Province, China; (2) relationship between population dynamics of Whitmania laevis and chemical factors in paddy fields of Guangzhou, China; (3) chemical environment of blood-sucking Hirudinaria manillensis in paddy fields of Guangzhou was described. The study also discussed the foraging, growth dynamics, life recycle and biology of reproduction of blood-sucking land-leech H. hainana and freshwater leech Hirudinaria manillensis, the taxonomy and the zoogeography. In addition, the morphology including histology and anatomy of the organic system of H. hainana was described. The Hirudinea can be classified into 2 subclasses (Branchiobdellidea and Euhirudinea), 3 orders (Branchiobdellida, Rhynchobdellida and Arhynchobdellida), 9 families, 33 genera and 111 species. The response of leeches to pesticides, including toxicity (LD50, LD95) of different pesticides to H. hainana, was presented. Medical usage of the leech Hirudin from salivary glands as anticoagulant material was discussed. Hirudin (from Hirudo medicinalis), Haemadin (from land-leech), and hementi (from Hementeria ghilianii) were identified and isolated.     

Progress in the study of ecology, zoogeography, group, control repellent and medical usage of Hirudinea in China

Hirudinea in China were reviewed. The ecological distribution of Hirudinea, the population dynamics of H. hainana, the adaptation of H. hainana to temperature, soil humidity and pH, the response of H. hainana to temperature, relative humidity and light, and the effects of human activities on H. hainana populations were discussed. The land-leeches' behavior ecology, including (1) the locomotion of H. hainana such as shortening, oscillating, rotating, sucking cleaning, rubbing and foraging behaviors; (2) the factors influencing these behaviors; (3) the ecological significance, was also discussed. The ecology of freshwater leeches including (1) behavior in paddy fields of Ninghsien, Zhejiang Province, China; (2) relationship between population dynamics of Whitmania laevis and chemical factors in paddy fields of Guangzhou, China; (3) chemical environment of blood-sucking Hirudinaria manillensis in paddy fields of Guangzhou was described. The study also discussed the foraging, growth dynamics, life recycle and biology of reproduction of blood-sucking land-leech H. hainana and freshwater leech Hirudinaria manillensis, the taxonomy and the zoogeography. In addition, the morphology including histology and anatomy of the organic system of H. hainana was described. The Hirudinea can be classified into 2 subclasses (Branchiobdellidea and Euhirudinea), 3 orders (Branchiobdellida, Rhynchobdellida and Arhynchobdellida), 9 families, 33 genera and 111 species. The response of leeches to pesticides, including toxicity (LD50, LD95) of different pesticides to H. hainana, was presented. Medical usage of the leech Hirudin from salivary glands as anticoagulant material was discussed. Hirudin (from Hirudo medicinalis), Haemadin (from land-leech), and hementi (from Hementeria ghilianii) were identified and isolated.     

中国蛭类动物生态地理类群特征及其防护医用研究进展

综述中国蛭类研究如下内容:(1)生态学.山蛭生态学,包括山蛭的生态分布,海南山蛭(H. hainana)的种群动态,对温度、土壤湿度和pH值的适应,以及对温、光、湿的综合反应和人类经济活动对其种群数量的影响;山蛭行为生态学,山蛭的运动包括慢缩短、快缩短、身体摆动和转动、洗刷运动、亲吻运动、觅食行为6程序,影响山蛭行为的一些因素以及对环境因素刺激的生态学意义;淡水水蛭生态学,包括浙江水田蛭类生活习性、广州水生光润金线蛭种群数量动态与水体化学因子关系、广州水田吸血菲牛蛭生活水体化学环境;山蛭和水生吸血菲牛蛭的觅食、生长动态、生命周期和生殖生物学;(2)形态学、分类学和动物地理学.形态学包括山蛭机能组织学、山蛭器官系统解剖;分类学,中国蛭类动物有2亚纲(蛭蚓亚纲、真蛭亚纲)、3目(蛭蚓目、吻蛭目、无吻目)、9科、33属、111种,占世界蛭类物种数约1/6;动物地理学,包括世界山蛭科属动物地理,中国山蛭科动物地理、中国医蛭科动物地理.(3)蛭类的防治和驱避,淡水吸血蛭类防治所用农药种类,不同农药对海南山蛭的毒力( LD50、LD95)及使用,并比较了12种驱避剂对海南山蛭的驱避效果.(4)蛭类的医学利用,蛭素是蛭类唾液腺分泌的一种抗凝物质,蛭素有水蛭素(Hirudin)、山蛭素(Haemadin)和吻蛭素(Hementin).记述了蛭素的分离、纯化和功能以及有关分子生物学内容.     

Primary structure and function of novel O-glycosylated hirudins from the leech Hirudinaria manillensis.

Hirudin from the leech Hirudo medicinalis is a most powerful anticoagulant, and many isoforms have been described. In the present work, the primary structure of two hirudins from the leech Hirudinaria manillensis has been elucidated. The antithrombotic activity is similar to that of H. medicinalis hirudins although the sequence identity is below 60%. Surprisingly, the hirudins were found to be glycosylated at one site. Sugar analysis after methanolysis yielded fucose, galactose, and N-acetylgalactosamine. These results combined with data from matrix-assisted laser desorption ionization mass spectrometry, plasma desorption mass spectrometry, capillary zone electrophoresis, and lectin-binding tests indicate that the sequence is Fuc-Gal beta 1-3GalNAc-(O-threonine). This structure shows an interesting similarity to human blood group H determinants.     

不同农药对海南山蛭毒力测定及其防治研究

在室内,利用速灭杀丁,叶蝉散,杀虫双,乐果,三氯杀螨,草甘膦等6种农药对海南山蛭（Haemadipsa hainane)进行毒力测定,比较各种农药LK50表明,速灭杀丁＞叶蝉散＞三氯杀螨醇＞杀虫双＞乐果,除草剂草苷膦对海南山蛭完全无毒杀作用,草甘膦＋速灭杀丁对海南山蛭仍有很强的毒杀作用,海南山蛭对速灭杀丁的敏感性为,成体＞幼体＞亚成体,在田间小区试验表明,1．5％速灭杀丁防治效果为95．08％,大面积应用表明“草苷膦药液＋0．5％速灭杀丁”防治效果最好,达94．74％,“草甘膦药液＋1％叶蝉散乳油”防治效果次之,达90．5％,草甘膦＋灭蛭农药是防治山蛭好药剂,达到灭蛭除草的目的。     

海南山蛭对土壤湿度和气温的适应

在室内以土壤湿度梯度作为海南山蛭Haemadipsa hainana的栖息试验表明,海南山蛭栖息于含水量的15.1％－16.77％水分的土壤表面;野外调查表明,海南岛橡胶林内海南山蛭栖息于16.54％－17.66％含水量的土壤表面;热带雨林者栖息于19.92%-25.73%含水量的土壤,室内实验表明,海南山蛭爬向30－35℃温度范围,占试验蛭数79.15％,在8－9℃不活动,10℃能山蛭活动,随温度升高活动蛭数增加,而15℃试验蛭答部活动,活动蛭数与温度关系为Y－18.6Z-172.3,R-0.976,P=0.01。在橡胶林内8－9℃山蛭不活动;10℃开始有山蛭活动,且随温度升高活动蛭数增加,当17℃时所有山蛭活动,活动蛭数与温度关系Y＝4.73X-3.97,R=0.93,P=0.1。首次使用土壤湿度梯度测定山蛭对土壤湿度的要求,这是一个新的有普遍意义的方法,并讨论了海南山蛭对土壤湿度气温的适应。     

Morphological and Genetic Variations of the Freshwater Leech,Hirudinaria spp., in Peninsular Malaysia

In this study the genetic diversity of local freshwater leeches (Hirudinaria spp.) was inferred using mtDNA COI gene analysis and compared with the gross external variations of 26 freshwater leech specimens obtained from the wild and leech farms. Based on a neighbor-joining tree generated from 516 COI base sequences, four distinct clades of Hirudinaria were seen with interspecific genetic divergence in the range of 7.6–14.5%. The external morphological variations based on the presence of stripes, location of gonopores, and anus separated the samples into four morphologically distinct groups matching the four clades obtained from the molecular data. Two black stripes at the ventral region were observed only in specimens found clustered with clades that contained the GenBank-reported H. manillensis, whereas the brown or dark green coloration without stripes on the ventral region was seen in samples that clustered with H. javanica and H. bpling clades.     

Study on foraging mechanism of leeches with different feeding habits based on chemoreception and foraging behavior

The leeches Whitmania pigra and Hirudo nipponia live in similar environments but have different feeding habits. At present, there are few studies of the foraging mechanism of leeches with different feeding habits. In this study, we first used maze tests to show that these two species of leeches could locate and distinguish their prey through chemosensory activity without mechanical stimulation. However, the two leech species have different foraging behaviors: Individuals of W. pigra move slowly and repeatedly adjust direction through probing and crawling to detect the location of prey (snails), whereas individuals of H. nipponia move quickly, and after determining the location of food (porcine blood), they quickly swim or crawl to the vicinity of their prey. Scanning electron microscopy (SEM) revealed that there are two types of sensory cilia and pore structures related to mucus secretion in the heads of both leeches. There are two differently sized types of chemoreceptors on the dorsal lip in W. pigra, which may have different functions during foraging, whereas in H. nipponia there is only one type of chemoreceptor, which is small. We detected the chemical components in the natural food of these two leech species by UHPLC–MS. There were 934 metabolites in the body fluid of snails and 751 metabolites in porcine serum; five metabolites unique to the body fluid of snails and to porcine serum were screened as candidate feeding attractants. Of these metabolites, betaine and arginine effectively attracted individuals of W. pigra and H. nipponia, respectively. In summary, leeches with different feeding habits use chemoreceptors to sense external chemical signals when foraging, and there are significant differences between species in foraging behavior, chemoreceptors, and attractants.     

Serotonin and Leech Feeding Behavior: Obligatory Neuromodulation

Leeches have distinct advantages for investigating the behavioralfunctions of monoamines. Serotonin (5-hydroxytryptamine, 5-HT)is synthesized by a population of identifiable neurons and storedat unusually high intracellular concentrations. Identified serotonergicneurons evoke biting, salivation and pharyngeal pumping, andneuronal 5-HT is required for these physiological componentsof leech feeding behavior. Those stimuli which initiate andterminate ingestion by intact leeches, excite and inhibit the5-HT neurons respectively. Hence, leech 5-HT neurons are activein contexts in which the animal feeds. In leeches representingdifferent orders of Hirudinea, homologous 5-HT neurons appearto have similar roles in feeding. Serotonin not only affectssalivation, biting and pharyngeal peristalsis in these annelids,but also in nematodes, arthropods and molluscs. These comparativedata suggest that serotonin may function generally in the modulationof feeding.     

Molecular Identification of Haemadipsa rjukjuana (Hirudiniformes: Haemadipsidae) in Gageo Island,Korea

There are 60 species of blood-feeding land leeches, 50 species belonging to the family Haemadipsidae and 10 species belonging to the family Xerobdellidae. Despite recent papers on the land leeches, their taxonomic identification is not fully understood, especially at a species level. In Korea, there have been no historical records of the terrestrial leeches, but recently an unrecorded blood-feeding land leech was discovered at Gageo-do (Island), Korea. Molecular analysis was used to identify the species of 29 leeches collected from Mt. Dock-Sil in Gageo-do. Conventional PCR was conducted using nuclear 18S rRNA and mitochondrial cytochrome c oxidase subunit 1 (CO1) genetic marker. The 18S rRNA sequences revealed that the leeches share 99.9% identity with Haemadipsa rjukjuana (inhabiting Taiwan), and the CO1 sequences revealed that the leeches are very close to H. rjukjuana (inhabiting Taiwan). The CO1 sequences were separated into 2 categories, 1 with 94.6% and the other with 94.3% similarity to the H. rjukjuana L00115A (inhabiting Taiwan). This new finding of the land leech is the first record in Korea. In addition, the north range of the distribution of the blood-feeding leech (Hirudiniformes: Haemadipisidae) should be reconsidered including Korea.     

体重、年龄和应激刺激对菲牛蛭繁殖性能的影响

采用单因素方差分析等方法探讨人工养殖条件下体重、年龄和环境应激对菲牛蛭繁殖性能的影响。结果表明,体重对卵茧大小和湿质量及幼蛭体长、体宽和湿体质量无显著影响(P>0.05),而亲蛭性腺系数、平均产茧数、孵化率和平均孵化数在各组间差异显著(P<0.05),其中体重10～15g组明显高于其他组(P<0.05);年龄对亲蛭平均产茧数、孵化率、平均孵化数和卵茧大小影响显著(P<0.05),其中2～3年龄亲本具有最高的平均产茧数、孵化率和平均孵化数,8月龄亲本所产卵茧个体明显大于其他年龄组(P<0.05),而卵茧湿质量及幼蛭体长、体宽和湿体质量在各年龄组间均无显著差异(P>0.05)。环境应激对亲蛭的平均产茧数、孵化率和平均孵化数影响显著(P<0.01),其中1～2次操作应激下的平均产茧数和孵化数最高,无操作刺激下的孵化率略高于1～2次操作刺激(P>0.05),而卵茧大小和湿质量及幼蛭体长、体宽和湿体质量在各应激处理组间无显著差异(P>0.05)。本研究获得了菲牛蛭繁殖的最佳体重和年龄,建议繁育生产中挑选10～15g和2～3年龄的个体作为亲本,期间进行1～2次操作刺激可提高其繁殖性能。     

A new species of mammalian trypanosome,Trypanosoma (Megatrypanum) bubalisi sp. nov., found in the freshwater leech Hirudinaria manillensis

Leeches have long been considered potential vectors for the aquatic lineage of trypanosomes, while bloodsucking insects are generally considered as the vectors for the terrestrial lineage of trypanosomes. The freshwater leech, Hirudinaria manillensis, is a widely distributed species in southern China and could potentially act as the vector for trypanosomes. Prior to this study, no trypanosomes had been reported from this leech. However, in this study, leeches were collected from three different places in Guangdong province, China, and a large number of flagellates were isolated and successfully cultured in vitro. Based on morphology, these flagellates looked like a typical trypanosome species. Analysis was carried out on the molecular sequences of the 18S rRNA gene and the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene. To our surprise, these flagellates were identified as likely to be a mammalian trypanosome belonging to the clade containing Trypanosoma (Megatrypanum) theileri but they are significantly different from the typical TthI and TthII stocks. Analyses of blood composition indicated that the source of the blood meal in these leeches was from the water buffalo (Bubalus bubalis). To further test if this flagellate from the freshwater leech was indeed a mammalian trypanosome, we transferred the trypanosomes cultured at 27–37 °C and they were able to successfully adapt to this mammalian body temperature, providing further supporting evidence. Due to the significant genetic differences from other related trypanosomes in the subgenus Megatrypanum, we propose that this flagellate, isolated from H. manillensis, is a new species and have named it Trypanosoma bubalisi. Our results indicate that freshwater leeches may be a potential vector of this new mammalian trypanosome.     

Phylogenetic relationships of fish leeches (Hirudinea, Piscicolidae) based on mitochondrial DNA sequences and morphological data

Utevsky, S. Y. & Trontelj, P. (2004). Phylogenetic relationships of fish leeches (Hirudinea, Piscicolidae) based on mitochondrial DNA sequences and morphological data. — Zoologica Scripta, 33 , 375–385.
Phylogenetic relationships of fish leeches (Piscicolidae) were deduced from combined mitochondrial DNA sequences of 12S rDNA and COI genes using Bayesian inference and Maximum Likelihood, as well as from a combined molecular-morphological data matrix using Maximum Parsimony. Monophyly of the family was confirmed. The traditional subdivision into three subfamilies, the Platybdellinae, Pontobdellinae, and Piscicolinae, received weak support, but was not challenged by alternative groupings. In contrast to prior classifications, a basal split emerged between the Pontobdellinae on the one hand, and the Platybdellinae and Piscicolinae on the other. The complex coelomic system of pontobdellines is viewed as plesiomorphic, and independent reductions of the coelom in other fish leech groups are hypothesized. According to the inferred phylogeny, seawater was the primary habitat of fish leeches. Eurasian freshwaters were colonized by a species-rich freshwater clade (genera Piscicola , Baicalobdella , Cystobranchus and Caspiobdella ) and, probably independently, by the Asian genus Limnotrachelobdella .     

Reproductive Behaviour and Parental Care of Helobdella striata (Hirudinea,Glossiphoniidae): a Leech that Feeds its Young

The leech Helobdella striata Kutschera (1985) is a protandrous hermaphrodite. It copulates by hypodermic injection: a spermatophore is attached to the skin of the partner and sperm injected through it. During their male phase, the leeches copulate repeatedly and with different partners. In the female phase, 3 to 7 cocoons, each containing 2 to 20 eggs, are produced from the female gonopore and attached to the ventral side of the parent. The larvae which hatch from these eggs cling to the ventral side of the parent. After they have used up their yolk supply and have developed into juvenile leeches they remain attached to the parent for a further three weeks. Helobdella striata capture Tubifex worms and give them to the young they carry. This access to food allows the juveniles to grow from a size of 1 mm to 6 mm length while being carried and protected by the parent. H. striata reared in isolation produce young; at present it is not known whether they reproduce by parthenogenesis or by self-fertilization. The probable phylogenetic development of parental care patterns in the Hirudinea is reviewed.     

The complete amino acid sequence of a hirudin variant from the leechHirudinaria manillensis

Unlike the European leechHirudo medicinalis, the Asian jawed leechHirudinaria manillensis is specialized for feeding on mammalian blood. In the salivary glands of both these leeches, there is a potent inhibitor of thrombin, called hirudin, which acts as an anticoagulant. We have reported previously the isolation and purification of a variant of hirudin, called bufrudin, from the head portions ofHirudinaria. In the present study, the complete amino acid sequence of bufrudin was determined by automated Edman degradation of peptide fragments generated after cleavage of protein with trypsin or thermolysin. Comparison of the primary structure of bufrudin, with hirudin HV1, show about 70% sequence identity with deletion of two amino acids, but the key amino acids at the C-terminus, involved in the inhibition of thrombin, are conserved. However, similar sequence comparison of bufrudin with hirullin P18, a hirudin variant isolated from the same leech species but from whole leech, instead of heads, reveals even less sequence identity of about 60%. From the amino acid sequence, it is suggested that the conformation of the C-terminal portion of bufrudin may be significantly different from hirullin P18, but similar to hirudin HV1, upon its interaction with thrombin. These results indicate that, as withHirudo leech, various isoforms of hirudin also exist inHirudinaria leech, with a significant change occurring in the structure of the molecule during the evolution of leeches.     

The complete amino acid sequence of a hirudin variant from the leechHirudinaria manillensis

Unlike the European leechHirudo medicinalis, the Asian jawed leechHirudinaria manillensis is specialized for feeding on mammalian blood. In the salivary glands of both these leeches, there is a potent inhibitor of thrombin, called hirudin, which acts as an anticoagulant. We have reported previously the isolation and purification of a variant of hirudin, called bufrudin, from the head portions ofHirudinaria. In the present study, the complete amino acid sequence of bufrudin was determined by automated Edman degradation of peptide fragments generated after cleavage of protein with trypsin or thermolysin. Comparison of the primary structure of bufrudin, with hirudin HV1, show about 70% sequence identity with deletion of two amino acids, but the key amino acids at the C-terminus, involved in the inhibition of thrombin, are conserved. However, similar sequence comparison of bufrudin with hirullin P18, a hirudin variant isolated from the same leech species but from whole leech, instead of heads, reveals even less sequence identity of about 60%. From the amino acid sequence, it is suggested that the conformation of the C-terminal portion of bufrudin may be significantly different from hirullin P18, but similar to hirudin HV1, upon its interaction with thrombin. These results indicate that, as withHirudo leech, various isoforms of hirudin also exist inHirudinaria leech, with a significant change occurring in the structure of the molecule during the evolution of leeches.     

Modification of leech behavior following foraging for artificial blood

In this study we examined whether the foraging for artificial blood affected the behavioral responsiveness of leeches to electrical stimulation of the body wall. After foraging for artificial blood, electrical stimulation of the posterior end of the leech significantly increased the percentage of stimulation trials that elicited locomotory activity—swimming and crawling—compared to the behaviors elicited when leeches did not forage or foraged for normal saline. On the other hand, shortening always dominated the behavioral profile of the leech to anterior stimulation even after foraging for artificial blood. In intact anterior end-isolated nerve cord preparations, we also found that application of artificial blood to the intact anterior end was sufficient to modify motor responsiveness to DP nerve stimulation. Full strength artificial blood had an overall negative effect on the likelihood of DP nerve stimulation initiating swimming and on the average length of elicited swim episodes compared to when pond water surrounded the anterior end. Application of a 10% solution of artificial blood to the anterior end led to an increase in the likelihood of DP nerve stimulation eliciting swimming.     

The results of a study of the species composition of fish parasites in the river basins of northeastern European Russia. Leeches (Hirudinea), mollusks (Mollusca), crayfish (Crustacea) and arachnids (Arachnida)

The report contains the results of faunistic study of parasitic Hirudinea, Mollusca, Crustacea and Arachnida occurring on fishes in the region of the S. Dvina, Mezen' and Pechora river basins. The parasite fauna in the first basin is represented by 3 species of leeches, 4 species of molluscs, 13 species of crustaceans and 1 species of water mites, in the second basin--4, 1, 11, 0; in the third basin--2, 7, 7, 1 species respectively. Total number of parasites in the region investigated: 4 leech species, 5 molluscs species, 16 crustacean species and 1 water mite species.     

海南山蛭种群数量动态与气象因素关系研究

在海南岛橡胶林内,每月观测海南山蛭Ｈａｅｍａｄｉｐｓａ ｈａｉｎａｎａ种群数量Ｂａ,用逐步回归分析方法研究了１０个气象因子对海南山６种数量的影响,结果表明,１）每年海南山蛭种群数量不同,６ａ间影响海南山蛭种群数量的主要气候因素是Ｘ１（月雨量）、Ｘ３（月雨日）和Ｘ５（月有露日数）;２）海南岛５～１０月份为雨季,海南山蛭这种群数量明显增大,影响山蛭种群数量的主要气候因素是Ｘ５和Ｘ１６（月最大风速和）和     

水蛭捕食两栖类卵

许多卵生动物具有对付食卵者的精巧策略以降低死亡率,因为早期发育阶段的捕食压力能显著影响具复杂生活史动物的生存。两栖动物卵被水蛭等很多物种所捕食。我们查阅了水蛭捕食两栖类卵的文献,发现27篇论文。在所报道的水蛭捕食两栖类卵的例子中,无尾类(3.6%,n=591)明显多于有尾类(1.6%,n=255)。此外,我们第一次记录到水蛭捕食四趾螈(Salamandrina perspicillata)卵,这也是西欧有尾类的第一例报道。我们没有发现两个两栖动物类群间存在差异。然而,当我们分别分析来自北美、欧洲和亚洲三个地区的数据时,发现北美的有尾类和无尾类间存在显著差异。水蛭可捕食各生活史阶段的两栖动物,因此,水蛭应归为一些两栖类的天敌。