Anti-leech activity of Scutellaria baicalensis and Morinda citrifolia extracts against Piscicola geometra

Piscicola geometra leeches are naturally infecting cobia juvenile. The leeches attach to cobia by sucking and biting its surface and provide the gate of second infection. Water extracts of Scutellaria baicalensis root and Morinda citrifolia leaves were used to be tested through In Vitro method to look for the anti-leeches activity against Piscicola geometra. In this study, a total number of 800 leeches from infected cobia were prepared. The anti-leech activity from water extract of S. baicalensis root and M. citrifolia leaves were compared in different dilutions of plant extracts for 96 hours. Significant anti-leech activity was observed with M. citrifolia leaves with 80% mortality of leeches. S. baicalensis root showed higher anti-leech activity with 100% mortality of leeches. The average time was needed for S.baicalensis root to paralyzing and kill the leeches were 8h, 40h, 48h, 72h, and 96h in various dilutions of S. baicalensis root. This study indicated that S. baicalensis water extract had a potent for new anti-leeches agent.


Introduction
Cobia (Rachycentron canadum) culture has become popular in more than 23 countries in the Asia-Pacific region [1]. However, rapid expansion and intensification of cobia culture has resulted in increasing incidences of disease outbreaks [2]. Parasitic diseases are particularly common to infect the gastrointestinal tract, gills, and skin of cobia, which have caused economic losses [3,4]. Moreover, leeches infestation are considered as the portal for other pathogens that develop secondary infection and lead to death [5].
It has been demonstrated that the impact of leeches are correlated with the level of infestation [6]. P. geometra is a member of Piscicolidae family which is considered as blood-sucking marine leeches [7]. It has a cylindrical body shape and an anterior bell-shaped sucker - [8]. Moreover, it has much higher rate of oxygen consumption, it prefers running water and it is oligotrophic with pH range varies from 7.0 to 9.0 [9]. Since the leeches are found in wide variety of habitats, it is conceivable that the leeches can tolerate much broader ranges of conditions [10]. P. geometra was previously reported to be found in Pleuronectes platessa and Myoxocephalus [11], Rutilus rutilus, Scardinius erytropythalmus, Blica bjoerkna, Tinca tinca [12], Esox lucius, Barbus rajanorum, Salmo salar, Salmo girdneri, Rutilus rutilus [13], Gasterosteus aculeatus, Cottus scorpius and Platichtys flesus [14].  Medicinal plants contain bioactive compounds and shows impact in controlling fish diseases as a low-cost and eco-friendly method and it has a broad spectrum of activity [15,16]. Scutellaria baicalensis and Morinda citrifolia have been widely used as medicinal plant [17,18]. unique compounds contained in those plants are for example baicalein, wogonin, and oroxylin that are highly present in the root of S. Baicalensis and they can directly bind with the active site of enzymes to increase the stimulation of protein synthesis [19]. S. baicalensis root has been reported to be useful to treat liver problems [20], diarrhea, hypertension, hemorrhaging, inflammations, and respiratory infections, as well as to act as antimicrobial, , anti toxoplasm [21] and anti trypanosomal activities [22]. The leaf of M. citrifolia has been studied for its antimicrobial, anticancer [23], antioxidant, and larvicidal activities [24,25]. Moreover, the plant extract has also been used as anti-helminthic and anti-parasitic agent [26][27][28][29]. The aim of this study was to determine the anti-leech activity of water extracts of S. baicalensis root and M. citrifolia leaves against Piscicola geometra.

Plant extract preparation
The dried roots of S. Baicalensis were purchased in the local market of Neipu, Pingtung, Taiwan. Fresh M. citrifolia leaves were collected from noni farm, Pingtung, Taiwan. The leaves were chopped into small pieces and rinsed with distilled water before put into the oven for 12 h. The dried plant was grounded separately in a mechanical grinder and stored in an airtight container. Water extract was prepared by boiling 150 mL distilled water with 10 g of plant powder at 95 ºC. After extraction, the supernatant was centrifuged at 4 ºC, 1000 g for 10 min. The clear supernatant was filtered and stored at -20 ºC.

Anti-leech assay
The clear supernatant of S. baicalensis roots and M. citrifolia leaves was diluted with sea water to obtain concentrations of 50x, 100x, 200x, 400x, 500x, 700 x, and 1000x dilutions.
Five leeches per petri dish were used in three replicates involving eight petri dish of seven different concentrations and a control (sea water). Their effects were screened for 96 h with 8 h interval for observation of mortality. The behavior of the leeches and the time used to paralyze and kill them were recorded. The dilutions were renewed every 12 h. The efficacy of the plant extract that were able to kill the leeches within 1-96 h reflects the anti-leech activities and would be used to treat infected cobia in the future. The differences between control and treated groups were analyzed by one-way ANOVA.

Results
The anti-leech activities of S. baicalensis root and M. citrifolia leave water extracts against P. geometra infestation are shown in table 1 and table 2

Behavior of leeches
Leeches (Piscicola geometra) use their sucker for locomotion; after the posterior sucker attached, they extend their bodies and the oral sucker will be attached ( figure 2A). The posterior sucker is drawn forward, it reattaches near the anterior sucker and it will be released ( Figure 2B). This movement is rapid and efficient. After detachment, the posterior sucker is applied high up on the ventral surface,

Discussion
Infestation of P. geometra has caused extensive damage characterized by small circular lesions with the presence of blood on the skin, eyes, and gill of cobia. The damaged scale is considered as the portal of bacteria and fungi infections that can lead to second infections [30]. Leeches are rarely pathogens to their host and only become a serious problem when the infestations are high [31].
No standard medicine has been registered to treat leeches infestation. Anti-histamine, calcium hydroxide, lidocaine, hypertonic saline, bicarbonate, and citrus extract are traditional treatments to treat leeches infection and have been noted as ineffective treatments [32]. Pharmaceutical plants can be used as a source for a wide variety of natural antioxidants and they have been proven as safe therapeutic effects to treat fish disease [33,34]. Flowering plant Scutellaria baicalensis and Morinda citrifolia have been listed in a traditional Chinese medicine (TCM) to treat many diseases [35]. The dried root of S. Baicalensis and dried leaves of M. citrifolia produce large amounts of bioactive flavones and provide a variety of specific health benefits [36,37].

Conclusions
This study has demonstrated that water extract of S.baicalensis dried root has stronger anti-leech activity than the water extract of M. citrifolia dried leaves and it has ability to inhibit the swimming activity of P. geometra. S. baicalensis water extract demonstrated an effective anti-leech activity against P. geometra infection. Further study on bath treatment of infected cobia and analysis of active compounds of S. baicalensis root extract will be required in the future.