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For Review OnlyHistopathological observation and health status of captive

Hippocampus barbouri Jordan & Richardson, 1908

Journal: Songklanakarin Journal of Science and Technology

Manuscript ID SJST-2019-0228.R2

Manuscript Type: Original Article

Date Submitted by the Author: 04-Nov-2019

Complete List of Authors: Senarat, Sinlapachai; Chulalongkorn University, Department of Marine Science, Faculty of Science

Keyword: Captive thorny seahorse, Histopathology, Kidney, Liver, Gill

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Songklanakarin Journal of Science and Technology SJST-2019-0228.R2 Senarat

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1 Histopathological observation and health status of captive Hippocampus barbouri

2 Jordan & Richardson, 1908

3

4 Thatpon Kamnurdnin1, Sinlapachai Senarat1*, Jes Kettratad1,2, Theerakamol Pengsakul3,

5 Wannee Jiraungkoorskul4, Ratree Sooksuwan5, Woranop Sukparangsi6,

6 Chanyut Sudtongkong7

7

8 1Department of Marine Science, Faculty of Science, Chulalongkorn University,

9 Bangkok, 10330, Thailand

10 2Center of Excellence for Marine Biotechnology (CEMB), Department of Marine

11 Science, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok, 10330

12 Thailand

13 3Faculty of Medical Technology, Prince of Songkla University, Songkhla, 90110,

14 Thailand

15 4Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400,

16 Thailand

17 5Phuket Marine Biological Center (PMBC), Department of Marine and Coastal

18 Resources, Phuket, 83120, Thailand

19 6Department of Biology, Faculty of Science, Burapha University, Chon Buri, 20131

20 Thailand

21 7Department of Marine Science, Faculty of Science and Fisheries Technology,

22 Rajamangala University of Technology Srivijaya, Trang Campus, Sikao, Trang, 92150

23 Thailand

24

25 *Corresponding autor: E-mail, [email protected]

26

27

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28 Abstract

29 Health status of the zebra-snout seahorse, Hippocampus barbouri Jordan &

30 Richardson, 1908 in captivity has been required for approval of its aquaculture

31 program. To precisely measure health status, histological observation can unravel the

32 problem to H. barbouri aquaculture and recently little is known about its histopathology

33 Hence, in this study we aimed to investigate histopathological appearance of three vital

34 organs including gill, kidney and liver in captive H. barbouri during its juvenile and

35 adult stages, by using histological techniques. In the juveniles from 14-days stage

36 (100% prevalence) towards 30-days adult stage (100% prevalence), the fish gill

37 exhibited intraepithelial edema and necrosis while hepatic tissue showed evidence of

38 intracytoplasmic vacuoles. In addition, histological alteration to renal tissues was

39 observed, including degeneration of renal tubules, rupture in the collecting tubules ,

40 necrosis, the presence of melanomacrophage and the infection of trematode parasites.

41 The parasites were found in 30-days adult fish stage in the kidney (33.3 % prevalence)

42 and in 60-days adult fish stage in the mesentery (33.3% percent prevalence). Taken

43 together, this study hightlights the health issue of H. barbouri in captivity, in particular

44 histopathological alterations in gill, liver and kidney, suggesting that aquaculture of this

45 seahorse still need better protocols for maintenance and preventing infection.

46

47 Keywords: Captive thorny seahorse, Histopathology, Kidney, Liver, Gill

48

49

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50 Introduction

51 The Zebra-snout Seahorse, Hippocampus barbouri Jordan & Richardson, 1908,

52 belonging to Family Syngnathidae, is one of the most important fishery resources in

53 Thailand. This seahorse species can be found along shallow shelter reefs, and algae and

54 seaglass beds in tropical and temperate zones (Foster & Vincent, 2004). In recent years,

55 overexploitation in particular overfishing and habitat deterioration are main causes of

56 dramatic decline in the H. barbouri population in Thailand (Personal communication).

57 In addition, H. barbouri is listed in the Appendix II of the Convention on International

58 Trade in Endangered Species of Wild Fauna and Flora (CITES) (Hutton & Dickson,

59 2000).

60 Due to decline in H. barbourin population, strategy and research has been

61 developing in order to gain successful aquaculture and captive breeding of this seahorse

62 in Thailand for seahorse conservation and this pioneer work was initiated by Phuket

63 Marine Biological Center (PMBC), Thailand under its Culture Project from 2011 until

64 the present (PMBC, Unpublished data). Indepth study of health status/lifestyle of the

65 seahorse through microanatomical reseach under captivity is required for understanding

66 environmental factors influencing its reproductive capacity and growth rate under

67 aquaculture development. Hence, in the present study, health status of captive H.

68 barbouri from the juvenile to adult stages was assessed using histopathological

69 assessment methodology.

70

71 Materials and methods

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72 Seahorse collection

73 The captive broodstock, Hippocampus barbouri were reared in a standard

74 culture system from the Phuket Marine Biological Center (PMBC) Thailand. During

75 juvenile stages (at 1 days, 5 days, 7 days, 14 days and 20 days) and adult stages (at 30

76 days and 60 days) (Kamnurdnin, 2017). Previous observation showed that the adult

77 state of H barbouri in captivity is considered as over a period of 30 days due to this

78 stage occurred the secondary growth development (Kamnurdnin, 2017). All seahorse

79 were considered to be a voucher speciemens deposited from Kamnurdnin (2017), which

80 were collected between October to December 2013 The detail of each stage is shown in

81 Table 1. Afterwards, they were acclimatized and maintained in mechanically circulated

82 and filtered seawater, constant aeration, a temperature ranging from 26-28 ˚C, a salinity

83 level of 31-33 ppt, and a photoperiod of 12:12 h light-dark. The H. barbouri were fed

84 with wild krill twice daily. The experimental protocol was approved by the Animal

85 Care and Use Committee of Faculty of Science in accordance with the guide for the

86 care and use of laboratory animal prepared by Chulalongkorn University (Protocol

87 Review No. 1423003).

88

89 Histological techniques

90 All fish samples were quickly euthanized by rapid cooling method (Wilson,

91 Bunte, & Carty, 2009). The external and internal anatomies were visually evaluated

92 under stereomicroscope and then, the whole fish were transferred to Davidson's

93 fixative. The samples were dissected in cross sections about 1 cm from head to tail and

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94 were processed using standard histological protocols (Presnell & Schreibman, 1997).

95 The paraffin blocks were sectioned at a thickness of 4 µm and stained with Harris’s

96 hematoxylin and eosin (H&E), Masson's Trichrome (MT) and periodic acid-schiff

97 (PAS) (Presnell & Schreibman, 1997; Suvarna, Layton, & Bancroft, 2013).

98 Histopathological alteration of the fish was examined and photographed under the light

99 microscope (Leica digital 750). Each histopathological alteration in the important

100 organs was calculated and present as percent prevalence (%).

101

102 Results and Discussion

103 Due to the lack of information about H. barbouri health under captive condition,

104 one requirement was to identify histopathological alterations of captive broodstock H.

105 barbouri for documentation and help in application to aquaculture development. This

106 method has been routinely recommended and used to assess the general health status of

107 fish under both laboratory and field studies conditions (Blazer, 2002; OECD, 2009;

108 Dietrich & Krieger, 2009). Here we observed histopathological conditions in several

109 vital organs through the histological techniques, as shown in Figures 1–3. In addition,

110 the prevalence of histopathological lesion is provided in Table 2.

111

112 Histology and histopathology of gills

113 Histology of the H. barbouri gill in normal condition with good health is shown

114 in Figure 1A. The structure of the four-gill filament (or non-respiratory filament) arises

115 from gill arch in a perpendicular manner, where they are parallel to each other. Each gill

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116 filament is histologically observed as double rows, which each filament is divided into

117 the primary lamellae and secondary lamellae [respiratory lamellae (Figure 1A)]. Only

118 intense lamellar epithelium lifting and intraepithelial edema of secondary gills were

119 clearly altered in juvenile at 14 days (100% prevalence, Figure 1B), 20 days (33%

120 prevalence) and adult at 30 days (100% prevalence, Figures 1C-1D). This indicates that

121 the gill may be a more specific and sensitive organ in H. barbouri. It was reported that

122 these lesions serve as a mechanism of defense by increasing the structural distance

123 between epithelium and pollution in low quality environment. This notion was

124 supported by several research regarding gill of teleost fish (Bury et al., 1998; Sola et al.,

125 1995), for example, Cyprinus carpino after exposed to 10 mg/L Thiamethoxam

126 (Georgieva, Stoyanova, Velcheva, & Yancheva, 2014), Acanthopagrus latus after

127 treated to HgCl2 concentrations (Hassaninezhad, Safahieh, Salamat, Savari, & Majd,

128 2014) and Poecilia reticulate exposed to chlorpyrifos (de Silva & Samayawardhena,

129 2002). Additionally, some reports have shown that the histopathological lesions on the

130 gills could be developed due to the sensitization of the membrane mucosal that lining

131 the lamellae with activity of mucous cells and chloride cells towards the water supplied

132 to the fishes (Boyd, De Vries, Eastman, & Pietra, 1980), which needs to be observed of

133 H. barbouri in further study. Although here we found a few lesions in the gill, this

134 histopathological conditions indicate the impaired gill in captive H. barbouri and reflect

135 its reduced physiological function of the gill (the respiration, excretory and

136 osmoregulatory functions)

137

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138 Histopathology of livers

139 Necrosis and intracytoplasmic vacuoles, also called “hepatocellular lipidosis”, in

140 juvenile liver occurred at 14-days adult stage and at 30-days adult stage (100 %

141 prevalnce) (Figures 1E-1F). The occurrence of the hepatocellular lipidosis and cellular

142 degeneration of hepatocytes was known to be related to liver damage, as well as the

143 consequent loss of liver function (Greenfield et al., 2008). A similar mechanism was

144 previously suggested by Hinton and Laure´n (1990), who postulated that a derangement

145 in lipid and protein metabolism (lipidosis) might play negative roles in abnormal

146 accumulations of triglycerides in hepatocytes. It was reported that the appearance of the

147 hepatocellular lipidosis is caused by several factors, for example, chlorinated

148 hydrocarbons and other contaminants (Hendricks, Meyers, & Shelton, 1984; Hinton et

149 al., 1992; Robertson & Bradley 1992; Schrank, Cormier, & Blazer, 1997), including

150 PCBs (Anderson et al., 2003; Teh, Adams, & Hinton, 1997). However, liver

151 histopathology is also related to old age, poor nutritional value of food sources, and/or

152 other environmental conditions (Hinton et al., 1992; Robertson & Bradley, 1992).

153 These idea was in accordance with liver histopatholgical observation from Sparus

154 aurata fed on the different dietary lipid contents (Caballero et al., 1999; Zilberg &

155 Munday, 2002). Therefore, based on hepatic histopathological condition of H. barbouri

156 under captivity, pollutant(s) contamination and other factors as above (Hinton &

157 Laure´n 1990) might play some parts in deteriorating heath of the sea horse in

158 aquaculture.

159

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160 Histopathology of kidenys

161 Kidney is one of the major organ affected from contaminated aquatic environment,

162 which can be visualized through histological observation. In this present study, both 14

163 and 30 months exhibited histological changes in the renal degeneration (100%

164 prevalence) throughout obvious melanomacrophage aggregates (100% prevalence,

165 Figures 2A-2B). These lesions were similar to histological damages of teleostean renal

166 tissues exposed to carbofuran (Yenchum, 2010), ammonia (Aysel et al., 2008), and

167 methyl mercury exposure (Melaa et al., 2007). This emphases the important function of

168 fish kidney for excretion of metal and hydrophilic pollutants as shown by Pritchard &

169 Bend (1984); Meinelt et al. (1997); Takashima and Hibya (1995). In addition, renal

170 tissues might be used for histophathological biomarker to observe the contamination of

171 pollutants in aquatic captivity. Further investigations are needed to accurately meaure

172 the metal and other chemical pollutants in captive water and the relation to histological

173 damages to renal tissue and functions.

174

175 Melanomacrophage centers (MMCs) and infection of trematode parasites

176 Melanomacrophage centers (MMCs), accumulation of pigmented phagocytes

177 functioning in phagocytosis of erythrocytes and infectious materials, play central roles

178 in several vertebrates’ immune system (Takashima & Hibya, 1995; Steinel & Bolnick,

179 2017). In teleost fishes, immune responses to parasites, pollutants as well as stress also

180 trigger the emergence of MMCs, which can be used as histological indicator for

181 pathogen infection (Alvarez-Pellitero et al., 2007; Sitja-Bobadilla, 2008). Here we

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182 observed MMCs at the mesentery in 30-days adult stage (33.3 % prevalence) and 60-

183 days adult stage (33.3% prevalence) (Figures 2A-2B). Interestingly, trematode parasites

184 were found only in 30-days and 60-days stages. This supports the notion that MMCs

185 occurred when fish hosts were infected with parasites. In addition, we observed that

186 location of MMC accumulation were not in close proximity to the parasites, which were

187 protected by thick cyst wall. It was possible that the presence of MMC was a good

188 indicator of general stress in this seahorse. This was in agreement with previous

189 observations, which can be very useful for those studying the health status of fishes

190 either in wild or captivity (Steinel & Bolnick, 2017; Tsujii & Seno, 1990). Also, the

191 infectious location of the parasites were different between the fish stages: in kidney

192 tissue at 30-days stage (33.3 % prevalence, Figures 2C-2D) and in mesentery at 60-days

193 stage (33.3% percent prevalence, Figures 2E-2F). We found that the degeneration of

194 renal tubules in the kidney was also found near the trematode parasites infection (Figure

195 3).

196

197 Conclusion

198 In this study, we provided evidence of various histopathological observations in

199 several vital organs from the zebra-snout seahorse, H. barbourin under captivity,

200 including edema of gill, hepatocellular lipidosis and kidney degeneration and infection

201 of parasites. In addition, different stages of development exhibit distinct prevalence of

202 diseases. From our findings, we proposed that the presence of hislopathological lesions

203 here reflect unhealthy status of the some seahorse stages under captive culture. We need

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204 to successfully keep the seahorse fish in captivity. Regular monitoring of water quality

205 and providing optimal nutrition for animals in aquaculture for H. barbouri is required

206 and should be continuously assessed by the Phuket biological center (PMBC), Thailand

207 in order to gain successful captive breeding of this species.

208

209 Acknowledgements

210 We would like to special thanks to all staffs of Phuket Marine Biological Center

211 (PMBC), Thailand and the members of the Fish Biology and Aquatic Health

212 Assessment Laboratory (FBA-LAB), Deaprtment of Marine Sceince, Chulalongkorn

213 University, Aquatic toxicology Unite, Department of Pathobiology, Faculty of Science,

214 Mahidol University, Thailand for their help in the laboratory. We also specially thank

215 Dr. David V. Furman for reading the grammatical structure in this manuscript.

216

217 References

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1 Figure Legands

2 Figure 1 Light photomicrograph of gill histology (A) with consisting of primary

3 lamella (PI) and secondary lamella (SI) of Hippocampus barbouri in cativity.

4 Lamellar epithelium lifting (L) and edema (Ed) of secondary lamellar of gills

5 at 14 day (B) and 2 months (C-D) and intracytoplasmic vacuoles (Iv) were

6 observed in the liver. Note: periodic acid-Schiff (A); Harris's haematoxylin and

7 eosin; (B, D, E); Masson's Trichrome (C)

8 Figure 2 Light photomicrograph of melanomacrophase centers (MMCs) and trematode

9 parasites of Hippocampus barbouri in cativity. A-B: MMCs at 30 days. C-D:.

10 Trematode parasites (Tp) at 30 days in kidney tissue. E-F: trematode parasites

11 (Tp) at 60 days in mesentery. Note: A-F: Harris's haematoxylin and eosin.

12 Figure 3 2 Light photomicrograph of the degeneration of renal tubule (squre) of

13 Hippocampus barbouri in cativity. It was a representative figure at 30 days.

14 Note: periodic acid-schiff

15

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16

17 Figure 1

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18

19 Figure 2

20

21

22

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24

25 Figure 3

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1 Table 1 Details of size and number of samples in Hippocampus barbouri in captivity

Seahorse stages Days Numbers Total length

(cm)

1 3 15.6±0.78

5 3 21.7±3.94

7 3 29±3.22

14 3 36̀±2.75

Juveniles

20 3 42.9±2.96

30 3 57.3±3.75Adults

60 3 65.4±2.45

2

3

4

5

6

7

8

9

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11 Table 2 Prevalence alteration (%) of histopathological observations in Hippocampus

12 barbouri in cativity

Tissues

(n = 3)

DaysLesions

Percent prevalence

Gills 14 100

20

lamellar epithelium lifting

and intraepithelial edema of

secondary gills

33.3

Liver 14 100

20

lipidosis and cellular

degeneration of hepatocyte

Kideny 14 100

20

Renal degeneration, rupture

in the collecting tubules and

necrosis

Kideny 30 MMC and Trematode

parasites

33.3

Mesentary 60 MMC and Trematode

parasites

33.3

13

14

15

16

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