*J. Song1,3, M. Nagae2, Y. Takao2 & K. Soyano1 Effect of Temperature Changes and EDCs on Reproductive Activity of Japanese Common Goby (Acanthogobius flavimanus) *J. Song1,3, M. Nagae2, Y. Takao2 & K. Soyano1 1Institute for East China Sea Research, Nagasaki University 2Graduate School of Fisheries Science and Environmental Studies, Nagasaki University 3Deakin University Good afternoon ladies and gentlemen. I am Jing Song, from Nagasaki University in Japan. It is my honor to stand here today and present my five years of research to all of you! In Japan, I am studying the effect of temperature changes and EDCs on reproductive activity of the Japanese common goby.

Global Warming Industrial pollution of Mersey Estuary, UK http://blogs-images.forbes.com http://www.google.com.hk Industrial pollution of Mersey Estuary, UK As we know, increased human activity is resulting in global warming, including a concerning trend of increasing global temperatures over the past several years. Furthermore, the pollution of important waterways has and still is occurring all around the world. For example, the industrial pollution of Mersey estuary in the UK. Additionally, EDCs pollution has been a focus for water pollution in Idaho. Therefore, increasing global temperatures and pollutants such as EDCs entering our waterways could cause an adverse effect on aquatic organisms including fish? http://www.google.com.hk EDCs pollution has been focused on Idaho waters pollution https://www.google.com.hk

Background Low High Water Temperature Changes Drive Early European Flounder Drive Early Spawning Migration (Sims et al., 2004) Red Seabream Impairs ovarian activity & Gene expression in brain (Okuzawa and Gen, 2013) Studies shown that low water temperature can cause the European flounder to start spawning migration earlier. In contrast, high water temperature can impact the ovarian activity of Red seabream and effect gene expression in brain. Water Temperature Changes

? EDCs: Endocrine Disrupting Chemicals EEs: Environmental Estrogens Sewage Agriculture Industry ? So how do EDCs effect fish health? Sewage produced from human activities such as, agriculture, industry and domestic uses, discharges artificial chemicals into waterways. Some of these chemicals mimic the functions of hormones, and effect fish lifecycles, including reproductive activity and egg development. We call them EDCs, Endocrine Disrupting Chemicals.

Reproductive Endocrine System VTG Liver or Ovary Testis Environmental Stimulation 11-KT Pituitary GnRH GTH FSH LH Endocrine activity is important in maintaining the reproductive system in fish. The primary structure of this system steams from the brain to the pituitary, gonad and liver. Two main pathways form the base of the endocrine system, the feedback and negative feedback loops. These control the homeostasis of different reproductive hormones including, GnRH, GTH (include FSH & LH), and Estradiol (E2) or 11-KT. An important hormone involved in reproduction is E2, its role is to stimulate the liver to produce VTG (a precursor of yolk protein for gonadal development), the blood then transports VTG to gonad for vitellogenesis. Gonadotropin releasing hormone (GnRH) Gonadotropin (GTH) Follicle stimulating hormone (FSH) 17 b-Estradiol (E2) Luteinizing hormone (LH) Vitellogenin (VTG)

? × : ? ? Background EDCs Medaka Mangrove Killifish Grey Mullet Effect Gonadal Development & Sex Ratio (Aoki et al., 2011) Medaka Disrupt Reproductive Activity (Mills and Chichester, 2005) www.websbook.com ? Previous studies have shown that EDCs can effect the gonadal development and sex ratio of grey mullet and disrupt the reproduction of medaka. Also, EDCs can modulate the gonadotroping subnit genes expression of mangrove killifish. However, there is little information on the mechanisms in which EDCs effect fish life cycles and how temperature can add to these effects. Mangrove Killifish Modulate Expression of Gonadotropin Subunit Genes (Rhee et al., 2010)

? ・ How about fish conditions, currently? Field survey (2012.7-2013.3) Natural factor EDCs Impact Global Warming ? ・　How about fish conditions, currently? Good model fish for estrogenic impacts and environmental changes survey ※ Widely distribute ※ Can be reared in artificial conditions ※ Living in coastal areas, easy to collect Japanese Common goby (Acanthogobius flavimanus) It is important to know the current fish condition in coastal waters off the coast of Japan, to determine how and if EDCs and temperature effect fish.

Exp1. Field Survey Tokyo Nagasaki Tsukuda-bori Yokojuke Taira River (2012.7-2013.3) Exp1. Field Survey Nagasaki Tokyo Taira River Tsukuda-bori Yokojuke 2012-2013 Jul. - March 2012-2014 Aug. & Oct. Sampling commenced in 2012, in the Taira River on the coast of Nagasaki and two additional sites in Tokyo, Tsukuda-bori and Yokojuke Rivers.

♂ ♀ Methods & Materials & Fishing Sampling Measuring Field survey (2012.7-2013.3) Methods & Materials Fishing Sampling Measuring Histological observation Gonad Blood & VTG ELISA E2 ELISA ♂ ♀ & Female and male fish were collected by line? fishing. Gonad was collected to observe the fish’s developmental stage. ELISA method was used to measure the plasma level of E2 (a sexual hormone) and VTG (an important protein for gonad development). In addition, the gene expression of VTG mRNA was also analysed. Liver VTG mRNA Expression

Nagasaki （Taira） Field survey ・VTG & E2 much higher from January. (2012.7-2013.3) Low temperature Nagasaki （Taira） Increasing photoperiod (VTG) (E2) A: Female B: Male Firstly, we checked the parameters from fish collected at the clean Taira River. It showed that, VTG and E2 values fluctuate, in male fish VTG and E2 are at low levels, however in females they increased in January to February. Histological data displayed vitellogenesis in fish from November to December, verifying VTG and E2 values as they show the same pattern. We checked the temperature and photoperiod changes in our sampling area, and found low temperature and increasing photoperiod would be the important factors causing the higher levels of VTG and E2 in female fish. It is interesting to note: male fish developing from August to October, with all male fish matured by November. In contrast, female fish just start vitellogenesis in November. Suggesting male fish mature early compared to females. ・VTG & E2 much higher from January. ・Male maturation is earlier than female. 10/28

E2 level is very high in male fish collected from Tokyo Field survey (2012.7-2013.3) E2 level is very high in male fish collected from Tokyo Nagasaki : Taira Tokyo Bay：Yokojyukken, Tsukudabori Water quality at sampling sites How about fish living in contaminated sites in the two Tokyo rivers? The data is shown here. Compared to male fish living in Taira River, male fish living in the two Tokyo sites displayed E2 values 1000 times higher. Why did this huge difference happen? In order to explain the difference we obtained water quality data from these two sampling sites. The total EDCs detected in Tokyo waters is 3-4 times higher than in Nagasaki. Therefore, the increased levels EDCs detected could accumulated in fish over a long term period, causing the high level of E2 in fish sampled from the two Tokyo rivers.

Total estrogenic activity (E2-equivalent, ng/L) Field survey (2012.7-2013.3) Concentration of natural estrogens and alkylphenols in environmental waters at each site Sampling site Sampling date Total estrogenic activity (E2-equivalent, ng/L) Natural estrogens (ng/L) Alkyllphenols (ng/L) E1 E2 4-t-OP 4-NP BPA Tokyo Yokojuken 2012 1.54 5.72 1.22 16.49 2009.82 40.52 2013 1.02 5.85 0.66 288.19 1143.9 31.27 2014 0.94 7.86 0.58 8.26 414.33 269.96 Tsukudabori 1.60 4.49 1.34 114.05 1048.9 45.65 1.74 11.57 33.61 132.55 37.21 0.83 6.0 0.54 25.61 731.57 243.01 Nagasaki 0.21 0.27 0.2 10.41 5.89 4.68 0.45 1.07 0.4 3.55 41.55 13.92 1.39 0.13 5.04 53.24 2422.68 This table displays details of the EDCs in clean and contaminant waters. Clearly, total estrogenic activity is much higher in Tokyo compared to Nagasaki. Although, there are kinds of EDCs that occur naturally in the environment and some of them are present in high levels, estrogenic activity is mostly contributed to E2.

VTG mRNA Expression (copies /μg Total RNA) Field survey (2012.7-2013.3) (VTG) VTG concentration (ng/ml) Tokyo Nagasaki Yokojuken Tsukudabori Taira Tokyo Nagasaki Yokojuken Tsukudabori Taira Female Male VTG mRNA Expression (copies /μg Total RNA) Aug Oct Aug Oct Aug Oct Aug Oct Aug Oct Aug Oct 2012 2014 2012 2014 2012 2014 2013 2013 2013 N.D. (VTG mRNA) So with the high accumulation of E2 in fish, what happens to VTG production? We detected the VTG value in plasma and VTG gene expression in liver. It clearly shows that, much higher VTG production occurred in fish living in contaminated areas. Furthermore, when high expression of VTG gene was detected in fish collect in Tokyo, it was not detected in clean area. This indicates that high concentrations of EDCs in the water column can accumulate in fish and mimic reproductive hormones inducing E2, resulting in earlier and higher VTG production. E2 value were much higher in contaminated area. EDCs accumulation induce VTG earlier & higher production. 13/28

Known: Unknown: In the natural environment: ・　The development of goby is controlled by low temperature. ・　In contaminated environments, EDCs accumulate in water induces abnormal VTG production. Unknown: Clearly, fish collected in the Taira River were effected by low temperatures during development. In addition, fish collected in contaminated environments had abnormal VTG production as a result of exposure to EDCs. So will climate change impact fish development? ・　Climate change impact fish development ? ? ・ What kind of water conditions induce abnormal development ? ?

EDCs concentration (ng/L) Exp2. EDCs Treatment 1 week treatment Control Low Middle High EDCs concentration (ng/L) 2013.9 E2 (Estradiol) 10 100 1000 2015.9 0.1 1 NP (Nonylphenol) In order to understand the effect of EDCs on fish. A one week treatment trial was conducted on goby, using E2 and NP. Four groups were treated, one control and three treated groups with low, medium and high concentrations of EDCs.

VTG mRNA expression Significant increase in High group (p<0.05) EDCs treatment (2013.9-2015.9) VTG mRNA expression R² = 0.06206 R² = 0.39032 No changes Significant increase in High group (p<0.05) conversion rate of 4-NP to E2-equivalent (ng/L) is 0.00032 vtg gene shown similar pattern as ERa gene, elevated expression in high E2 treatment, but no changes in NP treatment. This indicates that E2 has a strong effect on reproductive hormone production in fish. E2 is strongly impact on fish. NP concentrations of 1000 ng/L showed no changes, which is equal to 0.32 ng/L E2.

Exp 3. Temperature Effect Winter (2015.1~) Control Low High 20 oC 15 oC 25 oC Winter (2015.1~) 6 weeks temperature affect in vivo 15-11.7 oC Middle Low High 20oC 15 oC 25 oC 3 months temperature affect in vivo Autumn (2015.10~) Control In order to know the effect of high temperatures on fish an in vivo experiment was conducted. Fish were divided into four groups, one in control and three treated groups with three separate temperatures: 15, 20 and 25oC. In 2015 this experiment was conducted for 6 weeks in winter and repeated in autumn for 3 months, to determine the effect on maturing and immature fish. 24.2-17.5 oC

Autumn (2015.10~2016.1) (Immature Fish) Temperature effect (2015.1-2016.2) Autumn (2015.10~2016.1) (Immature Fish) ERb1 mRNA Expression 25˚C Initial 30 days 60 days 90 days E2 concentration (pg/ml) Decreased Tendency So what would happened when the same treatment was conducted on immature fish in Autumn? During the 3 month test period, natural temperatures declined to almost 10oC. The E2 value in control group did not change to much however, a slight positive trend was observed in 15 and 20 oC groups. In contrast, a clear negative trend was shown in the high temperature group. High temperature caused a decrease in E2 gene expression, especially during vitellogenesis !!! (P<0.05)

Conclusion Future research: ・ Abnormal E2 production occurs high EDCs accumulation in water above 10ng/L. ・　High temperature decreased E2 production in immature fish displaying their sensitivity to temperature. Future research: ・　EDCs target tissue of fish is brain, pituitary or liver? ? ・ Temperature affect on GtH. ? ? Clearly, abnormal E2 production occurs when fish exposed to E2 over 10ng/L. In addition, high temperature decreased E2 production in immature fish displaying their sensitivity to temperature. So what is the interaction between temperature and EDCs in fish?

Thank you for your attention! That’s all. Thank you very much!