根據前人的研究報告指出：外溫動物面臨外界壓力時會提昇代謝速率 以獲取額外能量面對逆境(Houde, 1989; Pörtner and Knust, 2007)，另外，
隨著胚胎發育時期的進行，頭足類中的歐洲烏賊(L. vulgaris)和歐洲虎 斑烏賊(S. officinalis 胚胎代謝能力與需求也有所增加(Pimentel et al., 2012)。為探討頭足類胚胎在發育時期面對高溫緊迫的環境下，其體 內氨基酸代謝的效能，我們測定經由氨基酸代謝後排放至卵鞘中 PVF 的銨離子(NH4+
)濃度(Hoeger et al., 1987)。根據實驗結果顯示：高溫緊 迫下胚胎各發育時期 PVF 中銨離子濃度都較控制組高 (Fig. 5)，顯示 胚胎面對高溫緊迫體內的代謝反應與能量需求會提升，以供調節體內 平衡與保護細胞等機制對抗外界壓力。然而，卵鞘厚度隨著胚胎發育 有逐漸減薄的現象，導致滲透度逐漸增高進而促進了 PVF 中銨離子
32
與外界環境的擴散作用(Rosa et al., 2012; Schmidt-Nielsen, 1997)，因此 在實驗結果中隨著胚胎發育其 PVF 中銨離子濃度逐漸下降(Fig. 5A)，
這現象並不代表胚胎發育時，氨基酸代謝的效能不斷下降，或是面對 外界環境時其細胞採用此代謝途徑的機制下降。也有如先前討論結果，
因長期處於高溫緊迫對於無脊椎變溫動物胚胎或幼體會造成發育不 全，甚至個體死亡(Rosa et al., 2012)，故發育晚期胚胎(hatching)實驗 結果不能進行準確推論。
除此之外，我們偵測生理調控中與細胞呼吸代謝相關的
cytochrome c oxidase subunit I (COX I)基因表現相，與細胞膜電位恆定 相關的 Na+/K+-ATPase (NKA)蛋白表現量發現：短期的高溫處理兩者 都出現明顯下降現象，而長期的高溫處理下卻有顯著上升趨勢(Fig.
6,7)。我們推測短期的高溫緊迫造成 COX I 與 NKA 表現量受到抑制 是因為細胞內的 ROS/RNS 的迅速累積，誘發了 HIF-1的活化，除了 可能促使細胞進行適應性能量移轉(adaptive metabolic shift)機制，同 時抑制粒線體電子傳遞鏈的活性，降低 ROS/RNS 的繼續累積(Taylor, 2008)，因此我們測定的電子傳遞鏈上 COX I 基因表現相才會出現下 降情況 (Fig. 6)；但這種短暫抑制 ROS/RNS 產生的負回饋生理機制 也會影響其他協助面對逆境的代謝途徑，使胚胎整體能量分配出現失 衡狀態，甚至對細胞造成危害。另外，我們檢測了運用主動運輸穩定
33
細胞膜電位的 NKA 蛋白，發現其表現量有明顯下降現象(Fig. 7)，我 們推測是因為細胞為了防止能量轉移分配不均的情況加劇，進而抑制 運用主動運輸耗能機制的離子通道，以降低細胞能量需求。
頭足類動物胚胎面臨短期高溫緊迫的生理應變機制能藉由
HIF-1的活化有效降低細胞的氧化壓力。然而，胚胎細胞在長期高溫 緊迫伴隨著發育成長時，個體的整體代謝率必然提升並需要更多的能 量供給，相對地會繼續造成細胞內代謝銨離子或 ROS/RNS 的累積，
細胞除了必需將這些有害的代謝產物排出，並且需要提升抗氧化因子 與抗逆境因子等保護機制以防止細胞傷害。在諸多生理機制需要穩定 的條件影響下我們發現：頭足類動物胚胎在高溫緊迫下因為細胞內 ROS/RNS 的含量能夠有效維持，因此導致 HIF-1在長期高溫緊迫下 已有下降趨勢(Fig. 8)，對電子傳遞鏈的抑制作用因此減弱，造成 COX I 基因表現相的結果顯著上升(Fig. 6)，並且促進整體代謝機制啟動以 提高能量獲取。另外，前人的研究報告指出：具有維持細胞膜電位恆 定的 NKA 與調控細胞內銨離子濃度有直接相關(Ip and Chew, 2010;
Randall et al., 1999)，因此長期高溫緊迫下所測定的 NKA 蛋白表現量 明顯上升(Fig. 7)，說明頭足類動物胚胎必須藉由活化 NKA 的蛋白表 現以協助排放在長期高溫緊迫下產生的銨離子進而維持胞內恆定。
34
結論
迴游性的萊氏擬烏賊的胚胎時期由於是附著於沿岸潮間帶礁石區域，
不能通過轉移棲地避免非生物性的環境緊迫壓力，因此可能促使萊氏 擬烏賊胚胎演化出適應環境的對策以提高存活機率。根據我們的研究 結果顯示：萊氏擬烏賊在胚胎發育時期確實具有良好的細胞抗氧化壓 力機制與合適的代謝策略轉移機制協同面對外界的高溫緊迫，可是其 生理上的演化速度能否追上近年人類對自然環境的影響，我們仍然存 疑。雖然本實驗中萊氏擬烏賊胚胎內的 ROS/RNS 在細胞中濃度並不 會因高溫緊迫而明顯提升 (Fig. 2)，然而在飼養的過程中我們發現：
發育初期的萊氏擬烏賊胚胎只要曾經接受高溫處理超過 28 小時後，
即使回復到正常溫度下馴養，胚胎晚期出現胚體畸形或死亡的情況甚 高，即使成功孵化出幼體，幼體出現早產徵狀而容易死亡的現象也十 分普遍。這充分說明萊氏擬烏賊胚胎面臨高溫壓力時的生理適應性代 謝策略並無法在每個發育時期完全有效地防止細胞毒害，其體內的代 謝與生理機制間的協同調適機制還需要進一步研究。
35
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40 Table 1 Primers used for qRT-PCR
F, forward primer; R, reverse primer
Protein name Abbreviation Primer sequence Amplicon
size (bp) Accession numbers Catalase CAT F 5’- TACAACGCCATTGCGAACGGAAAC -3’
R 5’- AAGTACCATTCGGCCAACAGGGAT -3’ 159 AEJ33812.1 Superoxide dismutase SOD F 5’- TGGAAAGATCGTGTCGTCGTGGAA -3’
R 5’- CGCTGTATGGTGATCGGATATCGT -3’ 90 HM157275.1 Peroxidase Prx F 5’- TGAGAGCAATGACAGGCGGTCTTA -3’
R 5’- AATGTTGGCCAGACGGTTGTGTTC -3’ 121 S77774.1 Glutathione peroxidase GPx F 5’- ACTGCACATCCACTTTGGGT -3’
R 5’- TGTGGACTGTAACGTTGGACTGGA -3’ 128 AY675197.1 Nitric oxide synthase a NOSa F 5’- TGGAAAGATCGTGTCGTCGTGGAA -3’
R 5’- ATCCATACCCAATCAGCCGGACAT -3’ 164 AY582749.1 cytochrome c oxidase I CoxI F 5’- ACCGAATTAGGTAAACCCGGCTCA -3’
R 5’- AGGGACAAGTCAGTTACCAAAGCC -3’ 138 AY131065.1 Heat shock protein 70 HSP70 F 5’- TCAACACCAAGTCAACGGTCGAAG -3’
R 5’- TGGTCAAGCCAGCTGATTACCTCT -3’ 109 HM157267.1 Heat shock protein 90 HSP90 F 5’- CCTGACGATGAGGAGTCTAA -3’
R 5’- CAACCTTCTCGACCTTCTTG -3’ 106 JN825731.1 Reference genes
Ubiquitin-conjugated enzyme UBC F 5’- ATGCAGATGGCAGTATTTGCCTGG -3’
R 5’- TTATTGGCTGGGCTGTTTGGGTTC -3’ 127 HM157280.1
Temperature
(℃) Egg mass (g)
Embryo + yolk mass
(mg)
Embryo mass (mg)
Total length (mm)
mantel length (mm)
yolk length (mm)
Stage-24
29 5.56 ± 0.41 117.90 ± 13.6 32.87 ± 9.17 34.49 ± 1.76 10.92 ± 1.14 17.53 ± 0.69 32 4.90 ± 0.67 119.63 ± 11.63 35.37 ± 9.57 33.33 ± 2.39 9.90 ± 1.16 17.00 ± 1.73
Stage-29
29 11.60 ± 1.30 202.27 ± 12.54 169.43 ± 12.60 44.56 ± 0.96 21.93 ± 0.93 11.62 ± 0.17 32 11.31 ± 0.62 175.50 ± 13.73 135.73 ± 6.81 43.77 ± 0.71 20.95 ± 1.01 11.57 ± 1.87
Stage- hatching
29 - - 158.70 ± 25.70 34.26 ± 1.24 22.46 ± 0.34 - 32 - - 167.50 ± 11.94 33.22 ± 1.82 21.90 ± 0.62 -
Table 2 The basis physiological measurement in Sepioteuthis lessoniana embryos (from stage 24, stage 29 and hatching) in respiration rate experiment at two different temperature：28℃ and 32℃.
41
Figure 1 Respiration rate (μmol O2 /h gEM ) in Sepioteuthis lessoniana embryos (A) (from stage 24 to hatching) and ammonium (NH4+) concentrations in
sampling water (B) at two different temperature：28 ℃ and 32 ℃. (one-way ANOVA, Tukey test, e.g a significant difference is observed between ‘a’ and ‘b’, but not between ‘a’, P<0.05)
-10
Re sp ir at ion r at e (
mo l O
2/h g
EM) in S ep ioteu th is les so n ian a em b ry o s
stage 24
Embryo development stage a
stage 24 stage 29 hatching
Embryo development stage
28
oC 32
oC
Ammonium
(
NH 4+)
conentration(
mol/g EM)
in sampling watera
B
42
0.0 0.5 1.0 1.5
25
oC 32
oC
DCF fluorescence (fold of control)
4 h (acute) 28 h (chronic)
Figure 2 The value of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in Sepioteuthis lessoniana embryos. Detection of various free radical species were using OxiSelectTM In Vitro ROS/RNS Assay Kit. Values were presented as mean ± standard deviation (SD) (n=8). *, Significantly different between control and hyperthermic group (Student’s t-test, p<0.05).
0.0 0.5 1.0 1.5
25
oC 32
oC
25oC 32oC
43
0 1 2 3 4
mRNA expression of hrp normalized by ubc
25oC 32oC 0
mRNA expression of sod normalized by ubc
*
mRNA expression of cat normalized by ubc
25oC 32oC 0.0
Figure 3 Effects of hyperthermic circumstance on anti-oxidants, catalase (cat), superoxide dismutase (sod) and horseradish peroxidase (hrp), expressions in Sepioteuthis lessoniana embryos. The mRNA expressions were detected by qRT-PCR. Values were normalized by ubiquitin-conjugated enzyme (ubc) and
presented as mean ± standard deviation (SD) (n=4~6). Significantly different between control and hyperthermic group (Student’s t-test, *, p<0.05, **, p<0.01)
A. Superoxide dismutase (sod)
B. Catalase (cat)
C. Horseradish peroxidase (hrp)
0.0 0.4 0.8 1.2
1.6
*
mRNA expression of hsp70 normalized by ubc
25oC
mRNA expression of hsp90 normalized by ubc
25oC 32oC
4 h (acute) 28 h (chronic)
Figure 4 Effects of hyperthermic circumstance on stress-resistant genes (Heat
Figure 4 Effects of hyperthermic circumstance on stress-resistant genes (Heat