Hirudo Leeches and Science

Optimization of Growth Conditions of Hirudinea sp.
May 04, 2020

Optimization of Growth Conditions of Hirudinea sp.

Australian Journal of Basic and Applied Sciences, 5(3): 268-275, 2011 ISSN 1991-8178

Optimization of Growth Conditions of Hirudinea sp.

Zulhisyam A.K,, A. Anwar Ismail and Ibrahim Che Omar

Department of Agro Industry, Faculty of Agro Industry and Natural Resources, Universiti Malaysia Kelantan, Pengkalan Chepa, 16100, Kota Bharu, Kelantan.

Abstract: Leeches (Phylum: Annelida, Class: Hirudinea) are widely distributed all over the world in various habitats, such as freshwater, seas, desert, and oases (Gouda, 2006). In this study, the effect of light intensity, temperature and diet on the reproductive efficiency of Hirudinea sp. was examined with eight different conditions. After 3 months of culture, the number of cocoons produced was very significantly different among the different conditions (p=0.00). The average number of hatchlings per cocoon was significantly different (0.05) where condition 1 gave the highest number (6.23±0.25), but hatching rate was not (p=0.354). The condition 5 produced the highest mortality of parent leeches (52±13.86%). The sizes of the cocoons were not significantly different among the treatments, with the condition 1 having the largest cocoon of 22.19±0.92mm and 13.26±0.07mm according to their length and diameter, respectively. The wet weight of cocoons was significantly different (0.05) with the condition 1 producing the heaviest cocoons of 1.26±0.11g compared to condition 5 producing the lightest cocoons of 0.22±0.38g. The effect of diet (FT1: fresh eel blood and FT2: booster) on the growth and survivorship of the juvenile leeches was also studied. After 2 months of culture, the final body weight was significantly different among the treatments, with juveniles in the FT1 (fresh eel blood) had the highest final body weight (0.8893±0.012g). Percentage weight gain (WG) and specific growth rate (SGR) of the juveniles in the treatment FT2 (booster) were lowest with mean and standard deviation of 769.41±11.54% and 3.6±0.02%, respectively. Juveniles in the FT2 (fresh eel blood) treatment had the highest survival rate (93.33±5.77%).

Key words: Hirudinea sp., temperature, light intensity, survivorship, reproduction, growth INTRODUCTION

Leeches are distributed all over the world in a variety of habitats; in freshwaters, seas, deserts, and oases. They are important components in food chains; as predators, vectors of parasites, preys of aquatic animals (Sawyer, 1986). They occur in habitats that range from terrestrial to aquatic (both marine and freshwater) environments and are found on all continents. Leech was used by toxicologists and pharmacologists as a convenient tool for various investigations (Mann, 1962; Herter, 1968; Sawyer, 1986) in the past when its natural resources were boundless. In recent years, some leech populations have declined dramatically due to over-exploitation for fishing bait and medicinal purposes (particularly in Europe and Asia), and due to pollution (Sawyer, 1981; Elliot and Tullett, 1984; Wells and Coombes, 1987; Petrauskiene, 2003; Trontelj and Utevsky, 2005).

Hirudinea sp. is a sanguivorous, freshwater leech, with a wide distribution in Southeast Asia, such as in southern China, the Philippines, Thailand, Vietnam and Malaysia. In Malaysia, these leeches are known as ‘Lintah Kerbau’ (406th Medical Lab. Special Report., 1968). Traditionally, leeches are widely used as a model animal in toxicological, physiological, neurobiological, biochemical, histological and many other studies (Mann, 1962; Flerov and Lapkina, 1976; Lapkina and Flerov, 1979; Sawyer, 1986; Lapkina, 1992; Huguet and Molinas, 1992, 1996; Blackshaw and Nicholls, 1995; Petrauskien, 2001). There has been an increasing harvest of this species for medical purposes in the 20th century (Steiner et al., 1990; Electricwala et al., 1993; Singhal and Davies, 1996) and so is in Malaysia. In this country, it is not known or proven conclusively that the locally named Buffalo Leech is not of H. manillensis. Local taxonomists have not been able to identify the species used those for medical purposes and would rather refer to its genus only as Hirudinea sp.

During the reproductive process, parent leeches secrete cocoons that protect and often nurture the developing eggs during the critical stages of early development (Sawyer et al., 1981; Yang, 1996). Components of the cocoons are released from specialized glands situated within the clitellar sex segments, forming a sheath

Corresponding Author: Zulhisyam A.K, Department of Agro Industry, Faculty of Agro Industry and Natural Resources, Universiti Malaysia Kelantan, Pengkalan Chepa, 16100, Kota Bharu, Kelantan.

Email: mrzulhisyam@yahoo.com

Tel: +6013-9115161

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Table 1: Different conditions tested on reproductive efficiency of leeches.

FT1
Eel blood Eel blood Eel blood Eel blood -
-
-
-

-------------------------------------- FT2 Low High

Aust. J. Basic & Appl. Sci., 5(3): 268-275, 2011

around the clitellum into which fertilized eggs are deposited. The cocoon membrane is then passed over the head and sealed at both ends forming “plugs” at either end (Mason et.al, 2004). Embryos are dependent upon cocoon fluid contained in hard-shelled cocoons, while embryos from membranous cocoons can develop independently of the cocoon ( Marotta and Shain, 2007).

The breeding of leeches for medical purposes has bright commercial potential and of late many entrepreneurs have embarked on the farming of leeches. To meet the demand from clinical use, Chinese traditional medicine and other scientific research, there has been growing interest in culturing and breeding leeches in many countries (Yang, 1996; Trontelj and Utevsky, 2005). Several factors determine leech distribution in freshwater environments such as availability of food organisms; nature of the substrate; depth of water; presence of water currents; size and nature of the body of water; hardness and pH; temperature of the water; dissolved oxygen; siltation and turbidity; and salinity (Sawyer 1986). However, no study has investigated the factors affecting growth and production of leeches in the country. Particularly lacking is the effect of water, temperature, dissolved oxygen, pH, and light intensity on growth conditions of these leeches bred in a farm as well as the feeding requirements. The aim of the present study was to test the interactive effects of different temperatures, light intensities and diet on the reproductive performance of this leech species. In addition, the effect of different diets on juvenile growth and survivor rates were also studied.

MATERIAL AND METHODS

Origin of Broodstock:

Hirudinea sp(Buffalo Leech) used in the study was provided by PT Dynamic Consultant Co., Kota Bharu, Kelantan. The leeches were cultured in concrete tanks (20 ×10 × 20 m) filled with non-chlorine water source which were from river, well and rain to a depth of 25 cm. The concrete tanks were divided to four compartments. Approximately 1000 leeches were cultured in every compartment. The water in the concrete tanks was not aerated and exposed to direct sun light. Water hyacinth was placed in the concrete tanks and the leeches were fed once on live eel blood every week and once with an artificial booster every month. Sand was placed in the concrete tank to a height of 12 cm. Before the start of the experiment, leeches were cultured for 1 week in an indoor aquarium filled with non-chlorine freshwater (30cm depth, 600L),aerated and 50% of the water changed once every 3 days. The temperature, pH and light intensity were maintained at 27.92 ± 6.62 °C, 6.8 ± 0.3 and 1000-1500 lx, respectively. Leeches were fed once on live eel blood and once with an artificial booster in the preceding week before the proper study was initiated.

Reproductive Performance or Efficiency:

The effect of temperature, light intensity and diet on reproductive efficiency of leeches was examined in a fully orthogonal experiment with three factors, each with two levels as presented in table 1.

Treatments

Condition/Level C1
C2
C3

CCCCC8

Feeding -----------------------------------------

Light Intensity (lx)

Temperature (°C) --------------------------------------- Low High
25-28 -
- 30-32
- 30-32
25-28 -
- 30-32
25-28 -
25-28 -
- 30-32

- 0 -
- 0 -
- - 100-150 - - 100-150 Booster - 100-150 Booster - 100-150 Booster 0 - Booster 0 -

Each treatment consisted of three replicates with a total of 24 aquarium tanks (30 x 19 x 26 cm) with 25 leeches in each replicate tanks. Approximately 600 leeches were collected from the holding tank, and randomly placed into the assigned experimental aquariums. Soil to about 25 cm depth was provided as substrate in each aquarium. Over the three months period (January 1, 2010 to March 31, 2010), daily observations were made.

Experimental Methods: Cocoon Deposition Number:

At the end of the experiment, all the cocoons deposited by the broodstock were collected and counted and the average deposition number of cocoons for each growth condition was calculated.

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Table 2: Ingredients of the booster experimental diet. Feeding treatment
Booster (FT2)

Methodology: Juvenile Growth:

Ingredients Compost
Molina
Ziolite Allobohpora rosea Phosphorus Calcium

Aust. J. Basic & Appl. Sci., 5(3): 268-275, 2011

Hatching Number and Hatching Rate:

After the juveniles were released from the cocoons, they were collected and counted. The ratio of the numbers of juveniles to deposited cocoons was calculated for each growth condition to obtain the average hatching number.

During the experiment, the abortion rate was high and the number of dead cocoons (juveniles failed to hatch) were counted and recorded. The ratio of dead to the deposited cocoons was then calculated as the average hatching rate for each treatment.

Broodstock Mortality:

The number of dead broodstock leeches was recorded during the daily observation.

Cocoon Size and Wet Weight:

All cocoons from each replicate were collected and their length and diameter measured. The wet weight of each cocoon was taken before hatching occurred.

Data Analysis:

Statistical analyses were conducted using the software SPSS 17.0 (Statistical Program for Social Sciences 17.0) to test the difference among the growth conditions and any differences obtained were considered significant at 0.05. The cocoon deposition number of broodstock leeches, hatching number and hatching rate of cocoon, survivorship of parent leeches, cocoon size and wet weight were analyzed by one-way ANOVA and where this effect was significant; Duncan test was performed to compare the treatments.

Effect of Diet on Juvenile Growth: Experimental Design:

Newly released juvenile leeches used in this experiment were cultured in the hatchery tanks for 3 days prior to the experiment. Two feeding treatments (FT1: fresh eel blood and FT2: booster) in three replicates per treatments with 20 juveniles per replicate were tested. The experiment lasted for 60 days. The indoor tanks used were the same size as that of the broodstock experiment (20 ×10 × 20m) and each was filled with non- chlorine water. The temperature, dissolved oxygen, pH, ammonia and light intensity were 27.34 ± 6.53 °C, 7.6 ±0.2 mg L-1, 7.4 ± 0.3 and 0.05 mg L-1, 1000-1500lx respectively. During the experiment, the juveniles were fed once every 10 days and the uneaten food was removed after each feeding. Any dead juveniles were removed and recorded daily. The ingredients of experimental diet FT2 (Booster) are shown in Table 2. The booster diet was mixed with soil with proportion of 1:4 (kg) before being fed to the juveniles. The FT1 (fresh eel blood) diet was fresh and placed directly into the tanks during feeding.

At the beginning of the experiment, 20 juveniles of each replicated were sampled to obtain the original wet body weight for each treatment. At the end of the treatment, the final body weight of juveniles in each treatment was determined again. The percentage weight gain (% WG) and specific growth rate (SGR) of the juveniles in each treatment was calculated using the following formulae:

% WG = 100 x (final body weight - initial body weight) / initial body weight (g) SGR = 100 x (ln final body weight – ln initial body weight) / 60 days

Juvenile Survivorship:

At the end of the experiment, the number of live juveniles in each treatment was counted and recorded, and then the survival rate was calculated.

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Reproductive parameters
Cocoon number (p=0.00)
Hatching number (p 
0.05) Hatching rate (%) (p 0.05) Cocoon length (mm) (p 0.05) Cocoon diameter (mm) (p =0.153) Cocoon wet weight (g) (p 0.05) Mortality rate (%) (p=0.00)

C1
6±1a# 6.23±0.25a 95.23±8.26a 22.19±0.92a 13.26±0.07a 1.26±0.11a 2.67±2.31a 

C2
4±1b 3.14±1.27b 62.77±25.64ab 19.27±0.19ab 11.49±0.13ab 1.05±0.01a 13.33±2.31a

C3 0.67±1.16c 0.5±0.87b 16.67±28.87b 5.58±9.67bc 3.39±5.87b 0.23±0.4b 44±4d

C4 1.33±0.58c 2.83±2.47b 70±51.96ab 16.6±0.39c 11.17±0.3ab 1.13±0.07a 33.3±2.3bc

C5 0.67±1.16c 2.17±0.29b 80±26.46ab 4.74±8.22c 3.34±5.78b 0.22±0.38b 52±13.86e

C6
0.67±0.58c
2.33±2.08b
66.67±57.74ab
9.48±8.21c
6.7±5.8ab
0.51±0.44ab
29.33±8.33b
different at the 5% of significant level as determined

Aust. J. Basic & Appl. Sci., 5(3): 268-275, 2011

Data Analysis:

Statistical analyses were conducted using the software SPSS 17.0 (Statistical Program for Social Sciences 17.0) to compare the means of two independent samples and any differences were considered significant at 0.05. The final weight gain, percentage growth rate, specific growth rate and survival rate were analyzed by independent sample t-test.

RESULTS AND DISCUSSION

Cocoon Deposition Number:

The number of cocoon deposited by the broodstock leeches was significantly different among the different of culture conditions (p=0.00). The average number of cocoons was highest in the Cwith mean and standard deviation of 6±1, followed by Cwith mean and standard deviation of 4±1. Both treatments differ significantly from the rest of the growth conditions of C3, C4, C5, C6, Cand Cwhich among themselves were not significantly different from one another (Table 3).

Hatching Number:

Hatching number was significantly different under different growth conditions (p 0.05). The condition under Chad the highest hatching number with mean and standard deviation of 6.23±0.25 while under the Cconditions the lowest hatching number was obtained with mean and standard deviation of 0.5±0.87. Hatching numbers of the cocoons in the C2, C4, C5, C6, Cand Cdid not differ significantly (Table 3).

Hatching rate:

Hatching rates of cocoons under different conditions were not significantly different (0.05). Although the Ctreatment had the highest hatching rate with value of mean and standard deviation of 95.23±8.26% but this was not significantly different among the eight conditions tested (Table 3).

Mortality of Broodstock Leeches:

Mortalities of broodstock leeches differed significantly under different growth conditions (p=0). The Chad the lowest mortality rate with mean and standard deviation of 2.67±2.31% compared with Cwhich gave the highest mortality rate of 52±13.86%. Mortality under other condition also showed a significant difference (Table 3).

Cocoon size:

The different temperature, light intensity and diet did not significantly influence the standard length and diameter of the cocoons produced (p$0.05, p=0.153). Cocoon standard length and diameter in the Cwere the largest with mean and standard deviation of 22.19±0.92 mm and 13.26±0.07 mm, respectively, whereas the cocoon standard length and diameter in the Ctreatment were the smallest with mean and standard deviation of 4.74 ± 8.22 mm and 3.34 ± 5.78 mm, respectively. There was no difference in cocoon standard length and diameter obtained those cultures in C2, C3, C4, C6, C7, and Cgrowing conditions (Table 3).

Cocoon Wet Weight:

The temperature, light intensity and diet imposed significantly influenced cocoon wet weight (p=0). Broodstock in the Ctreatment produced the heavier cocoons with mean and standard deviation of 1.26 ± 0.11 g, whereas the Ctreatment had the smallest cocoon wet weight with mean and standard deviation of 0.22 ± 0.38 g. Cocoon wet weight under the C3, C4, C6, Cand Cregimes did not differ significantly (Table 3).

Table 3: Comparison of reproductive features of the Hirudinea sp. under different of culture conditions: cocoon number, hatching number, hatching rate (%), mortality rate of parent leeches (%), cocoon standard length (mm), diameter (mm) and cocoon wet weight (g).

Culture conditions

C7
2±1c 1.67±1.53b 72.33±25.42ab 12.24±11.4c 7.68±6.79ab 0.76±0.67ab 37.33±2.31c

C8
1.67±1.16c 2±2b 74.33±36.14ab 9.41±8.15c 6.75±5.85ab 0.67±0.58ab 46.67±6.11de

#Data in the table were mean and standard deviation (mean ± S.D). Means with the same letter within the same column are not by Duncan test.

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Growth Parameters of Juvenile Leeches as Influenced by Diet:

Final body weight, weight gain, and specific growth rate of the juveniles were significantly different among the treatments (p=0). Final body weight of the juveniles when fed with fresh eel blood (FT1) was highest with mean and standard deviation of 0.8893±0.012 g and that fed with booster (FT2) was the lowest with mean and standard deviation of 0.665±0.004 g. Weight gain and specific growth rate under FT1 treatment was high, and in the FT2 treatment was the lowest with mean and standard deviation of 769.41±11.54 g and 3.6±0.02 g, respectively (Table 4).

Table 4: Effect of diet on the survivorship and growth performance of juveniles of Hirudinea sp. Diet

Growth parameters
Initial body weight (g) (
p=0.02)
Final body weight (g) (
p=0.00)
Weight gain (%) (
p=0.00)
Specific growth rate (
p=0.00)
Survivorship (%) (
p=0.04)
Data in the table were means and standard deviations (mean±SD.).

Influence of Diet on Juvenile Survivorship:

Booster (FT2) 0.07±0.00 0.665 ± 0.004 769.41 ±11.54 3.6 ± 0.02 71.67 ± 2.89

Fresh eel blood (FT1) 0.08±0.00
0.8893 ± 0.012 1016.29 ± 7.93

4.04 ± 0.03 93.33 ± 5.77

Survival rates of the juveniles under different treatments differed significantly (p=0.04). Juveniles when fed with fresh eel blood had a high survival rate of 93.33 ± 5.77 % when compared to those fed with booster (Tab

Discussion:

Determining optimum condition is a key factor for successful leech culture and reproduction. For example, mortality of the leech Hellobdella stagnalis is influenced by broodstock density and the density of their offspring (Mann, 1957). In this study, increasing temperature and light intensity had a negative effect on the number of cocoons that the broodstock produced. In general, under the condition where the temperature was 25-28°C with a light intensity of 0 lx and fed with a fresh eel blood cocoon deposition number was optimal depositing an average of 6 ± 1 cocoons per replicate. In the present study, it was found that each cocoon that was produced was laid on top of the soil (Fig.1). This number was lower within the range of that obtained with Haemadispa hainana (ranging from 4 to 8.15 cocoons deposited) (Tan et al., 1992), and that of N. obscura, (average of 8.33 ± 0.68 cocoons deposited) (Collins and Holmstrand, 1984). According to B. Zhang et al. (2008), increasing broodstock density had a negative effect on the number of cocoons that the broodstock produced. In his study, a density of 5 leeches per tank was optimal for cocoon deposition, with each leech depositing an average of 3.84±0.12 cocoons where the temperature and light intensity were 25.92±6.61 and 1000-1500 lx, respectively. The differences in the cocoon deposition number obtained in that study from the present one is that from the former it was derived from each leech in each treatment whilst in the latter study the number was based on 25 leeches per replicate treatment. B. Zhang et al., (2008) stated that the low cocoon numbers of broodstock leeches under high density appeared to be related to competition for food and space among the leeches, creating a stressful condition which directly affects the natural reproductive behavior. However, in the present study the effect of the broodstock density was eliminated by the use of the same density (25 leeches) in all cases.

The duration of leech growth, development and reproduction can often be different due to different culture temperatures (Tan et al., 1992). In the present study, the duration of reproduction was 102 days with a temperature range of 25-28 °C, which is within the normal range for growth and reproduction (19-32°C) for Hirudinaria manillensis (Yang, 1996; Tan et al., 2002). Hatching number for cocoons was significantly influenced by temperature, light intensity and diet, with the highest obtained (6.23±0.25) under the temperature regime of 27-28°C with light intensity 0 lx and diet fed with fresh eel blood. However, hatching rate was not significantly influenced as is shown in Table 4. In the study conducted by Tan et al. (1992) with H.hainana each cocoon produce 6-17 juveniles with a hatching rate of 77.4 %. In contrast, the present study showed that the growing condition with low temperature and low light intensity and fed with fresh eel blood gave the highest hatching rate with mean and standard deviation of 95.23 ± 8.26 %. Other than the inherent inter- specific differences between Hirudinea sp. and H. hainana, the differences in the culture preparation between Tan et al.’s study and this study may explain the higher number in Hirudinea sp.

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Fig. 1: Individual cocoon produced by an adult Hirudinea sp. laid on top of the soil 

In this study, the different treatments significantly influenced the size and wet weight of the cocoons that were produced, with the largest cocoons produced under the treatment which had low temperature, no light intensity and fed with fresh eel blood. Cocoon sizes in this study were approximately equal to the size (about 22 mm in mean length and 13 mm in mean diameter) reported by Tan et al. (2002). Generally, the wet weight of leech cocoons can be markedly different between leech species, e.g. 1.6-2.0 g in Whitmania pigra (Shi et al., 2006a) and 0.15-0.18 g in H.hainana (Tan et al., 1992). In this study, the wet weight of the cocoons ranged from 1.26±0.11 g under the low temperature, zero light intensity and fed with fresh eel blood to 0.22±0.38 g under the condition fed with booster at high temperature and high light intensity which gave the lowest wet weight of cocoon.

Water quality, temperature and parasitism are known to significantly influence the survivorship of the leech species (Sawyer, 1970; Tan, 2005; Shi et al., 2006b). Life-span of leech was also one of the key impediments for leech culture (Mann, 1957). In the present study, it was found that the different conditions of temperature, light intensity and diet could also markedly influence the survival rate of the parent leeches. A higher temperature and light intensity led to greater mortality as the growth condition was probably too extreme a result which was different with the study conducted by B. Zhang et al. (2008) where broodstock density had a significant influence on the survival rate of leeches. In this study, many leeches were found to be infected by parasitic protozoans and flatworms at the higher temperature and light intensity during the course of the experiment, which greatly influenced the survival and growth of the leeches (Fig.2).

Fig. 2: Dead leeches infected by parasitic protozoans and flatworms

Sawyer et al. (1981) and Peterson (1983) reported that newly hatched juveniles of the leech are independent of their parent once they emerge from the cocoons, and may show differential growth and survival rate in the wild due to diet (Tan et al., 1992). The juveniles of Hirudo orientalisH. verbena and H. medicinalis were reportedly consuming their first blood meal from amphibians whereas successive meals could

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be obtained from amphibians, fish or mammals (Sawyer, 1986; Keim, 1993; Graf et al., 2006). In this study, the final body weight of juvenile Hirudinea sp. fed with different diets were significantly different. Those fed with fresh eel blood (FT1) had the highest final juvenile body weight compared with booster (FT2) with means and standard deviations of 0.8893±0.012g and 0.665±0.004g, respectively (Table 4). During the course of the experiment, the juveniles in all the treatments were fed 6 times during the 60 days study in comparison to Tan et al. (2002) who fed the new-born juvenile of H.manillensis once during the 60 day culture. In their study, the final body weight of juveniles was only 0.27 g much lower that was obtained in this study. However, B. Zhang et al. (2008) reported a higher final body weight of juveniles of 0.98 g and 0.92 g fed with live bullfrog and fresh cattle blood, respectively, when compared with the present study although a different diet was used.

Diet composition can be important for growth and survival rates (Zapkuvene, 1972). In this study, it was determined that weight gain and specific growth rate of juvenile Hirudinea sp. fed with different diets were significantly different. The juveniles fed with fresh eel blood diet had the highest weight gain and specific growth rate with means and standard deviations of 1016.29 ± 7.93 % and 4.04 ±0.03, respectively. From the study conducted by B. Zhang et al. (2008) juveniles fed with live bullfrog had a highest weight gain and specific growth rate of 948.89 ± 35.76 % and 3.92 ± 0.06, respectively a result which was much lower when compared with the present study. The juvenile fed with fresh eel blood diet had the highest survival rate compared with those fed with booster during 60 days of culturing. B. Zhang et al. (2008) attributed that most of the juvenile leeches died mainly from hematemesis, probably due to their weak digestive system.

Conclusion and Recommendation:

This investigation has demonstrated that temperature, light intensity and diet significantly affect the reproductive efficiency of the leech, Hirudinea spA growth condition at a temperature of 25-28°C and zero light intensity fed with fresh eel blood is recommended for the commercial breeding of this species. The choice of diet is crucial on the growth and survival of the juvenile Hirudinea sp. where fresh eel blood is recommended as the diet in the first two months after cocoon release. 

ACKNOWLEDGEMENTS

The author is grateful to the Faculty of Agro Industry and Natural Resources for providing space and facilities to carry out the study. This study was funded by Universiti Malaysia Kelantan and supported by PT Dynamic Consultant Co., Kota Bharu, Kelantan and Freshwater Fisheries Research Centre, Gelami Lemi, Jelebu, Negeri Sembilan.

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Reproductive biology and ecological strategies of three species of medicinal Hirudo leeches
May 04, 2020

Reproductive biology and ecological strategies of three species of medicinal Hirudo leeches

Journal of Natural History

Vol. 45, Nos. 11–12, March 2011, 737–747

Reproductive biology and ecological strategies of three species of medicinal leeches (genus Hirudo)

Laima Petrauskiene ̇a, Olga Utevskaand Serge Utevskyc*

aInstitute of Ecology, Nature Research Centre, Vilnius, Lithuania; bDepartment of Genetics and Cytology, V.N. Karazin Kharkiv National University, Kharkiv, Ukraine; cDepartment of Zoology and Animal Ecology, V.N. Karazin Kharkiv National University, Kharkiv, Ukraine

(Received 9 May 2010; final version received 26 October 2010; printed 4 February 2011)

An analysis of the reproduction of three species of medicinal leeches (genus Hirudo) was carried out. The highest fecundity (34.34 ± 3.72 hatchlings per leech) was observed in H. verbana, the lowest in H. medicinalis (11.10 ± 2.56). The heav- iest hatchlings were found in H. medicinalis (0.046 ± 0.0005 g), the lightest in H. verbana (0.032 ± 0.0003 g). Hirudo orientalis had an intermediate fecundity (21.63 ± 3.39 hatchlings per leech) and its hatchlings were of intermediate weight (0.038 ± 0.0005 g). The species differ in their growth rate and mortality: Hirudo ver- bana had the smallest hatchlings and the lowest survivorship but its growth rate was highest among the three species. The results agree with sister relationships between H. medicinalis and H. orientalis and the basal position of H. verbana with respect to them. The reproductive traits of the three species suggest that H. verbana is an r-strategist that inhabits unstable environments, such as temporary ponds in steppe landscapes, whereas its congeners are subject to K-selection and occur in more stable and predictable habitats.

Keywords: Hirudo medicinalisHirudo verbanaHirudo orientalis; breeding; repro- ductive traits.

Introduction

The medicinal leeches (Hirudo spp.) have been applied in medicine for centuries (Phillips and Siddall 2009) and used as model organisms in various fields of the life sciences (Utevsky and Trontelj 2005; Petrauskiene ̇ 2008). During this long history, medicinal leeches were usually assigned to the species Hirudo medicinalis Linnaeus, 1758, but recently their taxonomic status has been revised dramatically. To date, it is generally accepted that four distinct species of the genus Hirudo – H. medicinalis Linnaeus, 1758, H. orientalis Utevsky and Trontelj 2005, H. verbana Carena, 1820 and H. troctina Johnston, 1810 – range through the western Palaearctic. Species sta- tus for these taxa has been corroborated by morphological differences (Nesemann and Neubert 1999; Utevsky and Trontelj 2005), random amplified polymorphic DNA molecular markers (Trontelj et al. 2004), nuclear and mitochondrial gene sequences (Trontelj and Utevsky 2005; Utevsky and Trontelj 2005) and both mitochondrial gene sequences and nuclear microsatellites (Siddall et al. 2007). These species also differ in their vicariant geographical distribution (Nesemann and Neubert 1999; Utevsky et al. 2010), biochemical composition of saliva (Baskova et al. 2008) and chromosome

*Corresponding author. Email: sutevsk@univer.kharkov.ua

ISSN 0022-2933 print/ISSN 1464-5262 online © 2011 Taylor & Francis
DOI: 10.1080/00222933.2010.535918 http://www.informaworld.com

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738 L. Petrauskiene ̇ et al.

numbers (Utevsky et al. 2009). The western Palaearctic species of Hirudo can inter- breed in aquaculture, but substantial reproductive barriers have been detected between them (Petrauskiene ̇ et al. 2009).

More recent investigations have revealed preferences of the four species of medic- inal leeches for different landscape types (Utevsky et al. 2010). Hirudo medicinalis occupies the deciduous arboreal zone of Europe. The range of H. verbana largely corresponds to the Mediterranean and sub-boreal steppe zones in Europe, Anatolia and Central Asia, whereas H. orientalis is associated with mountainous areas in the sub-boreal eremial zone and occurs in Transcaucasian countries, Iran and Central Asia. Hirudo troctina has been found in northwestern Africa and Spain in the Mediterranean zone.

The different environmental conditions suggest substantial eco-physiological dif- ferences between the species. Some autecological features of the medicinal leeches have been found. In her pioneering research, Zapkuviene ̇ (1972a) revealed distinct temper- ature optima for cocoon deposition in H. medicinalis and H. verbana (H. medicinalis f. serpentina and H. medicinalis f. officinalis respectively in the original publication). Hirudo medicinalis starts laying cocoons at temperatures 2lower than in H. verbana (Zapkuviene ̇ 1972a). Such a difference may lead to different phenologies in nature, e.g. an earlier start to reproduction of H. medicinalis in habitats with a sympatric occur- rence of H. medicinalis and H. verbana. When living in the same habitat (see Utevsky et al. 2010), the species may tend to be reproductively isolated by the different temper- ature optima in a kind of ecological isolation (see Coyne and Orr 2004). The length of the season favourable to reproduction and the type of water bodies (temporary or sta- tionary) are conditions that, at least in part, determine fecundity and juvenile growth rate. Zapkuviene ̇ (1972a) previously found some differences in fecundities and growth rates of H. medicinalis and H. verbana. Obviously, different environmental conditions lead to divergent biological properties of related species.

This research aims to reveal differences in fecundity, growth rate and survival rate in H. medicinalisH. orientalis and H. verbana and to correlate these with their ecological preferences.

Materials and methods

Samples

Fifty individuals of H. medicinalis, 40 individuals of H. verbana and 40 individu- als of H. orientalis were used in breeding experiments. All leeches were more than 14 months old, and were therefore mature (Zapkuviene ̇ 1972b). The H. medicinalis originated from a commercial aquaculture facility in Moscow (Russian Federation) and from ponds in the vicinity of Vasyshcheve (Kharkiv Region, Ukraine). The H. ori- entalis were purchased in a pharmacy in Kharkiv, although it is known that they came from Azerbaijan. Hirudo verbana were obtained from the commercial aqua- culture facility in Moscow; they were probably descended from leeches collected in the Krasnodar Territory (Russian Federation), which is a traditional area for the commercial harvesting of medicinal leeches (see Lukin 1976).

Other factors that can influence fecundity were excluded. It has long been known that the number of hatchlings per cocoon depends on the size of the maternal individ- ual (Sineva 1949). In our experiment, the average weights were 9.81 ± 0.62 g (range 3.05–19.36 g) for H. verbana, 10.33 ± 0.51 g (6.52–13.72 g) for H. medicinalis and

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Journal of Natural History 739

9.26 ± 0.83 g (4.00–18.72 g) for H. orientalis. An analysis of variance showed that there were no significant differences between the weights of the leeches in these three groups and as a result the impact of the size of maternal individual on fecundity was excluded.

Breeding

To explore fecundities and other biological traits of H. verbanaH. medicinalis and H. orientalis, breeding experiments were carried out. The leeches were kept and bred according to methods already used by several workers (Zapkuviene ̇ 1972a; Wilkin and Scofield 1991; Davies and McLoughlin 1996; Utevskaya 1998; Petrauskiene ̇ et al. 2009). All adult leeches were kept singly in 1-litre pots with dechlorinated water for 1 month before mating. After that, leeches were fed with bovine or porcine blood in sausage casings at 37C, and placed in pairs for copulation for 1 month more. The temperature was increased to 25C to stimulate copulation.

After the 1-month period for copulation, the leeches were kept individually in con- tainers with wet peat for another month for cocoon deposition. Cocoons were then collected from the peat. The cocoons were counted and the percentage of leeches that laid cocoons was calculated. Some leeches died before placing cocoons in the peat, some died in the peat, and so the percentage of fertile leeches was calculated from the number of leeches that survived in the peat. Every cocoon was placed into its own separate container, and containers were checked daily for 1 month to determine when hatchlings left cocoons. Hatchlings were counted for all mating pairs. If the young leeches did not escape from cocoons after 1 month, cocoons were opened and any live leeches found in these cocoons were counted as hatchlings as well.

Measurements

The weight of hatchlings was determined immediately after they left the cocoons and after each feeding. Five feedings were performed (at 2, 5, 11, 20 and 36 weeks after hatching). Hatchling weights were measured 2–3 weeks after feeding to provide leeches with an opportunity to remove water and salt ions associated with the blood meal. Medicinal leeches begin to concentrate ingested blood immediately after feed- ing (Boroffka 1968; Zerbst-Boroffka 1973; Wenning 1996); during the 2 weeks after the feeding period they lose 40–50% of their weight, but their weight subsequently stabilizes (Büsing et al. 1953; Zebe et al. 1986; Petrauskiene ̇ 2001, 2004). The leeches were fed with fresh bovine or porcine blood as described above. All groups received the same kind of blood at one time.

Dead hatchlings were counted every day and mortality was expressed as a percentage of the initial number of hatchlings.

Statistical methods

Differences between various experimental groups were determined using Mann– Whitney U-tests (central tendency comparisons) and F-tests (percentage compar- isons). As three groups were compared, the significance level for multiple comparisons (p) was 0.017 after Bonferroni adjustment (p′ p/0.05/0.017, where is the significance level for a single comparison, p′ is the significance level for multiple

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740 L. Petrauskiene ̇ et al.
comparisons, and is the number of pair comparisons). This method was used for

all traits.

Results

Fecundity

The results of breeding experiments are summarized in Table 1. The highest fecundity was found in H. verbana, the lowest in H. medicinalis.

The percentage of H. verbana that laid cocoons was 91.43%, which did not differ significantly from the percentage, 82.35%, found in H. orientalis. In H. medic- inalis this value was 37.50%, less than half as much as in the other two species and these differences (between H. medicinalis and the other two species) were significant (p0.017).

Other indices of reproductive ability – number of cocoons per leech that survived in the peat and number of hatchlings per cocoon – were also highest in H. verbana and lowest in H. medicinalis with significant (p0.017) differences between them. Hirudo orientalis and H. medicinalis were significantly (p0.017) different only in the number of hatchlings per cocoon.

The total number of hatchlings per leech that survived in the peat was highest in H. verbana and lowest in H. medicinalis, while H. orientalis had an intermediate value and all the differences were significant (p0.017). However, differences for number of cocoons and number of hatchlings calculated from leeches that deposited cocoons were insignificant.

Weight and growth

The weight of hatchlings was highest in H. medicinalis (0.046 ± 0.0005 g) and lowest in H. verbana (0.032 ± 0.0003 g). The weight of H. orientalis hatchlings was intermediate (0.038 ± 0.0005 g). The differences between all groups were significant (p0.017).

The three species had different types of hatchling weight distribution (Figure 1). The distribution of H. verbana had the maximum frequency towards the lower weights with a tail towards high values, whereas the distributions of H. medicinalis and H. orientalis were more symmetrical.

Juveniles received five blood meals over 9 months. Their weight increased and reached 2.47 ± 0.11 g in H. medicinalis, 2.55 ± 0.08 g in H. verbana and 2.74 ± 0.09 g in H. orientalis. Although mean hatchling weight of the three species varied significantly immediately after hatching, these differences were not significant after nine months. Small juveniles of H. verbana were similar in weight to those of H. medicinalis and H. orientalis.

The growth curves (Figure 2) demonstrate that H. medicinalis and H. orientalis had similar patterns of growth, whereas H. verbana initially lagged behind the other two species although its growth rate subsequently increased and caught up. After five blood meals, the body weight of H. medicinalis went up by a factor of 54, H. orientalis by 72 and H. verbana by 79.

Survival rate

Eight months after hatching, 11.06% of H. verbana leeches had died, 2.45% of H. ori- entalis had died and 7.71% of H. medicinalis had died. The survival curves of the three

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Journal of Natural History 741

Table 1. Reproductive traits of three Hirudo species.

Species

No. of leeches

% of leeches deposited cocoons

No. of cocoons per leech (all leeches)

No. of hatchlings per cocoon

No. of cocoons per leech (deposited cocoons)

No. of hatchlings per leech (all leeches)

No. of hatchlings per leech (deposited cocoons)

Hirudo verbana Hirudo orientalis Hirudo medicinalis

35 34 48

91.43 ± 4.73 82.35 ± 6.54 37.50 ± 6.99

3.29 ± 0.277 2.53 ± 0.356 1.65 ± 0.366

10.45 ± 0.710 8.55 ± 0.592 6.73 ± 0.434

3.59 ± 0.291 3.07 ± 0.393 4.39 ± 0.599

34.34 ± 3.72 21.63 ± 3.39 11.10 ± 2.56

36.25 ± 3.56 26.29 ± 4.14 31.29 ± 3.63

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742 L. Petrauskiene ̇ et al.

H. verbana

A

Weight, mg

H. orientails

B

Weight, mg

H. medicinalis

C

Weight, mg

Figure 1. The hatchling weight distribution of three Hirudo species. (A) Hirudo verbana; (B) Hirudo orientalis; (C) Hirudo medicinalis.

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Percent of individuals Percent of individuals Percent of individuals

Journal of Natural History 743

Number of meals

Figure 2. Growth curves of three Hirudo species demonstrating that H. medicinalis and H. orientalis had similar patterns of growth, whereas H. verbana initially lagged behind the other two species but its growth rate subsequently increased and caught up.

Hirudo species (Figure 3) demonstrate similar survival patterns in H. orientalis and H. medicinalis. The juveniles of H. verbana faced a high mortality for the first months after hatching.

The different rates of survival resulted in the highest total number of adult off- spring per leech that survived in the peat in H. verbana (30.54 ± 3.31) and the lowest number in H. medicinalis (10.24 ± 2.36), while H. orientalis had an intermediate value (21.10 ± 3.31). All differences were significant (p0.017). The numbers of adult off- spring per fertile leech were 33.41 ± 3.46, 27.33 ± 3.85 and 25.61 ± 3.65 in H. verbanaH. medicinalis and H. orientalis, respectively. These values did not differ significantly.

Discussion

Differences and similarities among Hirudo species

The breeding experiment demonstrated that Hirudo species differ in their reproduc- tive characteristics. Differences were found in many fecundity traits: the proportion of

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Weight, g

744 L. Petrauskiene ̇ et al.

Months after hatching

Figure 3. Survival rates of Hirudo species demonstrating similar survival patterns in H. orien- talis and H. medicinalis and a higher mortality in H. verbana.

leeches that laid cocoons, the number of cocoons, and the number of hatchlings cal- culated from individuals that survived in the peat. The highest fecundity was observed in H. verbana, the lowest in H. medicinalis. On the other hand, the heaviest hatch- lings were found in H. medicinalis, whereas H. verbana produced the smallest juveniles. Hirudo orientalis had intermediate fecundity, and its hatchlings were intermediate in size. The species also differed in their growth rate and mortality. Hirudo verbana had the smallest hatchlings and the lowest survivorship, but its growth rate was highest among the three species.

Two of the species, H. medicinalis and H. orientalis, bear a great resemblance to each other in a number of traits. They demonstrated similarities in distribution of hatchling weight and in growth and survival rates. This result agrees with the sister relationship of H. medicinalis and H. orientalis and the basal position of H. verbana

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Survival, %

Journal of Natural History 745

in relation to them as revealed by a phylogenetic analysis (Trontelj and Utevsky 2005; cf. Phillips and Siddall 2009). The same pattern of relationships was deduced from external morphology (Utevsky and Trontelj 2005) and biochemical composition of saliva (Baskova et al. 2008).

Life strategies of Hirudo species

The results demonstrate that H. verbana has the highest fecundity and juvenile mor- tality and small juvenile body size, whereas H. medicinalis and H. orientalis have the opposite characteristics. Though numbers of cocoons, hatchlings and adult off- spring calculated per fertile leech do not differ significantly, the percentage of fertile individuals is significantly lower in H. medicinalis in comparison with its congeners. This suggests greater selection for mate choice and lower tolerance to aquaculture conditions in H. medicinalis. The two distinct patterns of reproduction and post- embryonic development should be assigned to r-strategies and K-strategies, respec- tively. Hirudo verbana may be characterized as an r-strategist that inhabits unstable environments, such as temporary ponds in arid steppe landscapes (Utevsky et al. 2008, 2010). Its high fecundity and small juvenile body size are traits that have been shaped by r-selection. Conversely, H. medicinalis produces fewer juveniles with larger body sizes and lower mortality rates. Obviously, this species is subject to K-selection. Hirudo orientalis occupies an intermediate position between the two strategies or verges towards H. medicinalis. Both live in more stable and predictable environments including static ponds and lakes of the arboreal zone in Europe and mountainous landscapes of the Caucasus and Central Asia.

Different life strategies of the three Hirudo species may be responsible for asym- metric patterns of the reproductive isolation between them (see Petrauskiene ̇ et al. 2009). Hirudo verbana and H. orientalis were more fertile in interspecific crosses than H. medicinalis. Being fertilized by other species, H. verbana and H. orientalis showed a moderate decrease in fecundity, which was caused by the decrease of hybrid offspring fitness: low weight, slow growth and high mortality. The third species, H. medicinalis, had the lowest fecundity in the hybrid crosses. In crosses with H. verbanaH. medic- inalis did not yield adult offspring and in matings with H. orientalis the number of adult offspring was fewer than in intraspecific crosses by a factor of five.

We suppose that the asymmetric pattern of reproductive isolation may be caused by different life strategies of these species. As a K-strategist, H. medicinalis is more discriminating in mating and a possible loss in fitness is more substantial in the case of a wrong choice compared with species that produce more offspring. That explains why natural selection prevents the hybridization of H. medicinalis with individuals of other species.

On the other hand, H. verbana, as an r-strategist, has more numerous offspring. A possible loss in fitness would not be so critical in this species when mating with indi- viduals of another species compared with the K-selected H. medicinalis. Consequently, in H. verbana isolating barriers are not very strict.

The r-selected features of H. verbana suggest that this species is most suitable for breeding in aquaculture compared with other medicinal leeches, which are adapted to the specific conditions of their natural habitats. Indeed, recently it has been found that leeches marketed as H. medicinalis are actually H. verbana (Siddall et al. 2007). In spite of the fact that viable populations of H. medicinalis still persist in Europe, it

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746 L. Petrauskiene ̇ et al.

is H. verbana that is sold by commercial facilities and widely used in medicine as an effective remedy and in neurobiology as a model organism (Siddall et al. 2007) because of its ability to tolerate artificial conditions.

Acknowledgement

This research was supported by INTAS grant no. 05-1000008-8147 “The medicinal leech (Hirudo spp.), famous and unknown: taxonomy, conservation, and medical applications.”

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Medicinal leeches 'are the wrong kind'
October 11, 2018

Medicinal leeches 'are the wrong kind'

Scientists have discovered that a case of mistaken identity means that "medicinal leeches" which have been used by doctors for centuries are not officially approved for medicine.

As well as the doctors who use them today in plastic surgery, numerous researchers who have studied the medicinal leech have also been using a different kind of blood sucker from the one they thought, according to a DNA study that now calls their findings into question.

Hirudo medicinalis, described by the father of taxonomy Linnaeus in 1758, has long been considered the sole European medicinal leech. Yet, as early as 1827, at least five additional species were recognised. Now a study of their DNA has provided the first conclusive way to tell them apart.

DNA barcoding work led by Mark Siddall at the American Museum of Natural History has revealed that commercially available medicinal leeches, until now assumed to be the species Hirudo medicinalis, used around the world in research and after surgery, are actually a closely related but genetically distinct species, Hirudo verbana. Moreover, the study has shown that wild European medicinal leeches comprise at least three distinct species, not one.

Dr. Siddall and his colleagues analysed the DNA of wild leeches from across their range in Europe, as well as from samples supplied by commercial providers and university laboratories that use leeches to study the nervous system and the way genes regulate development.

Their analysis clearly showed that the commercial and laboratory specimens were not Hirudo medicinalis, as they were labelled, but rather Hirudo verbana.

This carries significant regulatory implications, said the team: Hirudo verbana has not been approved by the US Food and Drug Aministration - as was thought to have occurred in 2004 - and it has no special conservation status, unlike Hirudo medicinalis, which is still afforded protection under various conservation conventions.

Commercially available European medicinal leeches also are used extensively by biomedical researchers studying biological processes such as blood coagulation, developmental genetics, and neurobiology.

Studies of commercial specimens have figured prominently in the discovery and production of anticoagulants and so called protease inhibitors, some of which may have cancer-fighting properties.

That researchers have been mistakenly using Hirudo verbana in their work for decades may call much of this research, including hundreds of scientific publications, into question and force a reconsideration of what scientists think they know about this widely studied species. And they add that Hirudo verbana should also be offered protection.

But the discovery is not all bad news. "This raises the tantalising prospect of three times the number of anticoagulants, and three times as many biomedically important protease inhibitors as previously thought," said Dr Siddall.

"However, it will also require a more nuanced effort aimed at conserving these much-maligned animals, and in a manner that takes into account their impressive diversity."

Dr Siddall reported his discovery in the the Proceedings of the Royal Society B, with Peter Trontelj from the University of Ljublajana in Slovenia, Serge Utevsky from the V. N. Karazin Kharkiv National University in Ukraine, Tripp Macdonald of Rutgers University, and Mary Nkamany from the City University of New York.

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