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Response and transcriptomic analysis of new aquaculture species:

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Whitmania pigra Whitman (Annelida, Hirudinea) to the anesthetic

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tricaine (MS222)

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Abstract:

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The freshwater leech (Whitmania pigra Whitman) is an emerging aquaculture species

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with per-kilogram prices that exceed $40
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This study analyzed the effects of tricaine methanesulfonate (MS-

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222) on W
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The

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experiment determined the anesthesia and recovery times for seven concentrations of

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tricaine (0
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The anesthesia state of leeches was

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categorized into five normal stages: sedation, disequilibrium, anesthesia, and deep

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anesthesia
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During the

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experiment, the use of tricaine did not affect leech mortality
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In the control group, leech weight was reduced by 55
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10, and the average number of

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leeches hatched per cocoon was 27
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The anesthesia group had an average of 3
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85 hatching per cocoon, resulting in a 60
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Based on the experimental data, tricaine can be

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used as an anesthetic for long-distance transportation of pregnant leeches because it

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does not affect cocoon production
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Overall, this

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study established an anesthesia method to facilitate leech-related research and offered

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insight into the anesthetic mechanisms of leeches or even Annelida species
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Introduction

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In the aquaculture industry, anesthetics are commonly used for both experimental

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and practical purposes to reduce animal stress, minimize animal damage, and ensure

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animal survival
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In

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addition, many aquaculture practices violate animal ethical rules, raising concern

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among the general public and scientists [1]
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Clove oil, etomidate, quinaldine, tricaine methanesulphonate (MS-222),

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and 2-phenoxyethanol are the most commonly used anesthetics in aquaculture

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practices

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Administration (FDA), is the most commonly used in global fisheries and model

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animal experiments

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hydrophilic and suitable for submersion administration to aquatic animals
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Tricaine has been widely used to anesthetize fish and

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other aquatic animals in aquaculture operations
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mykiss), can produce stimulative effects such

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as erratic swimming and breaking the water surface

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necessary to determine the optimal anesthesia protocols for different aquatic species
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Tricaine, the only fish anesthetic approved by the U
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Food and Drug


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Studies


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W
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For thousands of years, the dried body of W
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At $40 per kilogram, a fresh leech is significantly more expensive

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than most traditional aquaculture species, such as crucian carp, prawns, and salmon
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When it comes to the aquaculture management of leeches, the

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long-distance transportation of pregnant leeches can easily result in damage and death,

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resulting in substantial economic losses for farmers
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Tricaine had a quick onset time and rapid drug

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metabolism, resulting in fewer drug residues

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anesthetic for W
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However, there was no published information regarding the

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anesthetic effects of tricaine on W
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[10]

, and it seemed to be a potential

[11]

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Therefore, the purpose of this study was to compare different concentrations of

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tricaine for anesthesia and recovery time in W
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In addition, we evaluated leech survival

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and spawning in the control and anesthesia groups
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2
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1 Animals

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W
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Regarding the leech culture system, the temperature

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remained around 25±1◦C
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Before beginning the experiments, leeches

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fasted for 24 h
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2 Effect of different tricaine concentrations

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The preparation method of the tricaine stock solution was as follows: In 0
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Tricaine stock solution was diluted to yield

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anesthetic solutions with different concentrations (0
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5 g/L, 1 g/L, 2 g/L, 3

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g/L, 4 g/L, and 5 g/L)
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The body weight

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range of experimental leeches was 9-11 g
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The anesthesia time began when W
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As soon as leeches were anesthetized with anesthetic solutions, they

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were immediately transferred to a recovery pool
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pigra entered the recovery pool and was recorded as it progressed through a different

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stage
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2
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pigra body weight on anesthesia

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Anesthesia time, recovery time, and survival rate were compared among three

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weight groups: the small weight group (10 ± 1g), the medium weight group (15 ± 1g),

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and the large weight group (20 ± 1g)
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The anesthesia and recovery duration were recorded

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according to the method described in 2
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In each group, five biological repetitions

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were performed three times
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4 Effect of tricaine on the spawning of W
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pigra, 90 small

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weight leeches were divided into the control and anesthesia groups
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In the anesthesia

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group, after entering the deep anesthesia period, leeches were placed in clear water for

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more than 24 h before being placed in the breeding tank to produce cocoons
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2
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pigra

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Six adult leeches were randomly assigned to one of two groups: the SZH group

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(not treated with tricaine) and the SZM group (treated with tricaine)
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For transcriptome analysis, libraries for each

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group (n = 3) were constructed using an equal amount of total RNA using the Vazyme

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RNA kit (Nanjing, China)
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A sample with clean data was mapped to the W
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Differential expression analysis was performed

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using R statistical package software EdgeR
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05 and Fold Change ≤ -2 or

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≥ 2 for down and up-regulated genes, respectively
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3 or r ≥ 0
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01) were annotated for biological processes (BP),

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cellular components (CC), and molecular function (MF) in the Gene Ontology (GO)

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database
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genome
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3
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1 Anesthetic stages division, recovery stages division, and the corresponding

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behavior characteristics

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After exposing W
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25

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to 5 g/L, its behavioral characteristic was observed
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pigra could be divided into five stages based on the standard grading of fish

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anesthesia and the observation results of this experiment, whereas its recovery process

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could be divided into two stages (Table 1)
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During the A3 stage, leech behavior included partial loss of equilibrium, erratic

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swimming, loss of sucker force, and over 120s flipping
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During the A5

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stage, leeches were unresponsive to external stimuli, lost complete muscle tone, and

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entered a deep anesthesia state ideal for transportation
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R1 partially restored the movement, and the suction

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force was still relatively weak
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3
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25 g/L,

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0
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pigra were investigated in this

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study
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Commonly used for fish and frogs, low concentrations of

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tricaine (0
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5 g/L) produce only slight sedation in W
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Within 22-39

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min of exposure to 1 g/L tricaine, leeches entered the A2 and A3 stages, but none

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reached the A4 and A5 stages
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The anesthesia time was significantly affected by anesthetic

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concentration
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The anesthesia-related videos

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have been deposited in file S1
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The recovery test results showed

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that increasing tricaine concentrations prolonged the recovery time of anesthetized

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leeches
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During the tricaine anesthetic experiments, no W
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3
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pigra on anesthesia

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Fig
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The times for the three weight groups of leeches to enter the A2 and A3

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anesthesia stages were almost identical (Fig
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The anesthesia times for the small

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and medium weight groups to enter the A4 stage were relatively similar, at 24
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94 min, respectively
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In the A5 stage, the large anesthesia weight

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group was 30
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21 and 42
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The large weight group recovered from the anesthetic state at the same anesthetic

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concentration significantly faster than the other two groups (Fig
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The recovery

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times for the small and medium weight groups were similar, and the recovery times

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for the small and medium weight groups at the R1 stage were 37
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30 min,

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respectively
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The large weight group recovered from the R1 and R2 stages

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in 21
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18 min, respectively, 15 min less than the other two groups
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3
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pigra

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A significant application of leech anesthesia was transporting pregnant leeches

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over great distances to the spawning site
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The outer layer of a leech is honeycomb-like or spongy yellow-

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brown; approximately 30 young leeches can hatch from each cocoon (Fig
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Before

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and after cocooning, leech weight decreased by 55
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While the weight of leeches before and after cocoon

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production decreased by 60
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50%
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10 cocoons on

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average, and 26
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In the anesthesia group, each

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leech produced 3
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35 leech

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larvae (Table 4)
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96-

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29
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67-19
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The long

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axis of the cocoons of the anesthesia group ranged from 12
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49 mm, and the

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short axis of the cocoons ranged from 9
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62 mm (Fig
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Compared to the

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anesthesia group, the cocoons of the control group were slightly larger
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2
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3
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On average, 80
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1% mapped to the reference gene, respectively
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52% of the reads mapped to CDS regions, 1
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33% of the reads mapped to intergenic regions, 7
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93% of the reads mapped to 3′UTR

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regions
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The heat map of the hierarchical clustering analysis revealed the

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significantly differential expressions between groups and indicated the high

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reproducibility of these DEGs (Fig S1)
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Most

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significantly up-regulated by anesthetic treatment were Sik2, Sik1, Girk3, Pcdh9, and

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Pcdh11x
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Anesthesia

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treatment caused one of the most notable downregulations of Pcdh1, which belonged

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to the protocadherin family
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The calcium ion-binding go-molecular function Ncald was

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significantly up-regulated
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The horizontal axis represented

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the enrichment degree, whereas the vertical axis represented the enriched GO Terms

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for the top 20 GO terms with the highest enrichment degree of up-regulated genes

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(Fig
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Ace showed significant enrichment in the Renin-angiotensin system, which

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regulated angiotensin-converting enzyme, according to the KEGG pathway analysis

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(Fig
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Both MAPK signaling pathway and phosphoinositide 3-kinase/ protein kinase

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B (P13K/AKT) signaling pathway were expressed differentially in the anesthetic

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group, including the up-regulated gene Nr4a2
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4
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pigra, the research object of this experiment, has crucial commercial and

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scientific value
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pigra is an emerging aquaculture species and also a crucial model

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for the study of neurophysiology, developmental, and regenerative biology

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rarity of anesthesia research on leeches hinders their culture and scientific research
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The stages of anesthesia in Largemouth Bronze Gudgeon Coreius

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guichenoti could be distinguished by several characteristic behaviors, such as loss of

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tactile sensation, opercular movement frequently, and swimming posture, among

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others [14]
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During the anesthetization of leeches, the swimming speed, the ability to

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maintain stability, the suction force of the suction cup, the ability to restore normal

[12-13]

[15]


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These

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posture after being flipped, and the responsiveness of leeches to external stimuli were

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the primary characteristics observed [16]
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pigra was divided into five stages (A1-Normal, A2-Sedation, A3-Disequilibrium, A4-

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Anesthesia, and A5-Deep anesthesia) and the recovery process was divided into two

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stages (R1 and R2)
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W
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Moreover, despite its strong toxicity and side effects, high

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tricaine concentrations can be used to euthanize fish

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comprehensive consideration, 2 g/L tricaine was recommended for sedation and

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transportation to avoid irreparable damage to leeches
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pigra was immersed in a tricaine solution with a

[18-19]


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pigra was

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significantly higher than that for fish (the general effective dose was 50-200mg/L),

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and the anesthesia and recovery times were significantly longer than those for fish
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pigra was at least 2 g/L, which is greater than 10

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times the effective dose in other aquatic animals
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There were three possible reasons: (1) Fish have a complete

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circulation system, which accelerates the arrival of the drug at the receptor site and

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facilitates its absorption, distribution, metabolism, and elimination
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(2) Leeches are a model organism for neural research because their nervous

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system is relatively finite and interconnected with relative simplicity
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Leeches lack a blood circulation system, which could explain why anesthetics are

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head ganglion, 21 mid-body ganglia, and a tail ganglion connected by two lateral

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connecting nerves and one smaller medial connecting nerve [23-24]
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Advanced nervous

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systems were conducive to effect generation and information transmission
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Leech skin was

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responsible for exchanging gases, ions, and water with the surrounding environment

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[10]

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in fish and leeches
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The anesthetic agent was dispersed in the


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pigra generally ranged from 10-20 g, and the

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reproductive performance of leeches increased significantly with mass

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the influence of leech body weight on the anesthetic effect of tricaine was studied

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using 10-20 g of leeches
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It was speculated that an increase in fish body weight could not

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result in a significant increase in fish gills area and that small body weight could be

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more conducive to anesthetic absorption and metabolism
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According to

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experimental results, tricaine had little effect on the spawning of leeches
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Multiple genes affecting

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signal transduction, neurotransmitter release, and sodium ion concentration were

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among the 29 differentially expressed genes in the SZM group (treated with tricaine),

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which may play a crucial role in the anesthetic tricaine mechanism
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Several

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genes, Sik1 and Sik2, were found in the SZM group
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It was speculated that the up-regulation of Ncald in the SZM group affected

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neurotransmitter release by regulating Ca2+ channels

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specific aspects of neuron function

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and causing an anesthesia effect
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The mechanism required further experimental research and analysis
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Three genes in the


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Since

[38]

, leading to changes in

[39-41]

, altering intracellular calcium concentration,

[42]


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pigra
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Tricaine is a safe anesthetic with minimal effects on the cocoon

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production of W
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pigra housekeeping

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genes, key signaling pathways, and physiological functions
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