探討環境熱壓力對花翅搖蚊幼蟲Chironomus kiiensis Tokunaga (Diptera: Chironomidae)的影響及恢復反應的研究

Abstract

探討環境壓力對生物的基因表現的反應是直接且快速，特別是針對短期的干擾例如環境熱的壓力。隨著全球暖化的影響，短期且高強度的熱浪是未來不可避免的氣候事件。其中，淡水生態系是支持生物生命及維持陸域健全生態系重要的資源之一，但其卻易受熱浪的影響，特別是淡水生態系中重要的水生昆蟲等對於水溫升高而的反應是非常巨大且脆弱的。前人的研究顯示水生昆蟲在持續熱壓力的環境下，反應出較小的體型且提早進入成熟期以增加繁殖的世代數。此外，物種組成的改變如優勢物種增加的反應也得以在前人的研究中發現。這些反應可能會直接或間接影響淡水生態系面對壓力後的永續性。搖蚊(Diptera: Chironomidae)是作為淡水生態系中最主要的族群之一，其適應環境壓力的能力可以做為評估淡水生態系中壓力所導致之不利影響的生態指標。因此，本研究目的在探討花翅搖蚊幼蟲Chironomus kiiensis Tokunaga (Diptera: Chironomidae)基因轉錄組於39 ˚C (ET) 及40 ˚C (HET) 的短期壓力(24h)下的適應能力。此外，測量形貌上的改變如體長、觸角長、大顎長、體寬等因子可提供更多的證據顯示形貌受到熱壓力的影響。本研究中花翅搖蚊對熱壓力的適應能力分成兩個層面進行探討：抗壓的反應(第二章節)及壓力恢復的反應(第三章節)。首先，實驗先將花翅搖蚊幼蟲分別暴露於兩個極端溫度(HET及ET)及控制組溫度(CT = 23 ˚C)維持24小時，並探討搖蚊對不同壓力強度的反應(詳見第二章)。接者，探討花翅搖蚊在熱壓力停止之後4小時(R4h)及24小時(R24h)的反應來評估花翅搖蚊面對熱壓力(HET及ET)之後恢復的能力(詳見第三章)。第二章的結果顯示花翅搖蚊面對24小時HET及ET的壓力下皆提升熱休克蛋白的基因表現已增加抗壓的能力。此外，HET影響下的花翅搖蚊幼蟲顯示出核酸代謝是最主要的反應代表HET導致了細胞的破壞。HET的壓力也造成細胞中能量匱乏導致存活下來之個體體型明顯較ET及CT小。相較於HET，花翅搖蚊面對ET表現出很高的適應能力，對能量缺乏及體型改變的影響低。 第三章的結果顯示花翅搖蚊面對HET及ET產生的反應是可恢復的,包括恢復熱休克蛋白的基因表現(HET於R24h恢復、ET於R4h恢復)。HET造成的能量匱乏及細胞破壞也在R24h有恢復的表現。此外，細胞修復的機制於HET恢復的過程中是重要的，於R24h有核醣體生合成作用(ribosomal biogenesis)的反應發生。雖然本研究結果顯示花翅搖蚊可以從HET的壓力恢復，然而於恢復過程中為了優先進行細胞的修復需要大量的能量導致體型成長的犧牲。從形貌特徵上可觀察到於R24h下從HET恢復並存活下來的幼蟲及前蛹個體有較小的體型，包括較短的體長及觸角以及較小的體寬，並降低了幼蟲化蛹的成功率。 整體而言，本研究的結果顯示花翅搖蚊對24小時HET及ET的壓力表現出很高的適應能力，包括提升抗壓力及壓力恢復的反應。然而，本研究結果顯示從39˚C提升至40 ˚C的1 ˚C之間發生關鍵的改變，導致存活下來的個體較小，這指出了水生昆蟲對於面臨未來高強的的氣候事件的脆弱性。

Changes in genetic expression of organisms are direct and rapid responses to stress, in particular, short-term disturbances such as thermal stress. As the global warming effects, short-term and high intense heatwaves are inevitable events in the future climate. Freshwater is one of the most essential resources for sustaining life of organisms and supporting health ecosystems, and will be profoundly affected by heatwaves particularly those aquatic insects which are considerable and vulnerable to warming temperature. Under the ongoing thermal stress, smaller body sizes, earlier emergence, increased voltinisms, or increased dominance of tolerant taxa have been documented for several aquatic insects in previous studies. These responses may directly or indirectly affect the sustainability of freshwater ecosystems after stress. As the one of the dominant aquatic taxon in freshwater habitats, the acclimation capacity of aquatic midges (Diptera: Chironomidae) can be an important indicator to evaluate the likelihood of adverse ecological effects on freshwater ecosystems caused by thermal stresses. Therefore, we aimed to study the acclimation capacity of Chironomus kiiensis Tokunaga (Diptera: Chironomidae) in response to two levels of short-term (24h) extreme thermal stress at 39˚C (ET) and 40 ˚C (HET) using transcriptome analysis in this study. Also, the morphological characteristics including body length, antennal length, mandible length, and body width of experimental individuals were measured to provide visible evidences on thermal effects. In this study, the acclimation capacity of C. kiiensis were discussed in two aspects, stress resistance (chapter 2) and stress recovery (chapter 3). Firstly, the experimental larvae were exposed to two extreme temperature, i.e. HET and ET, and control (CT at 23 ˚C) for 24h to investigate the thermal responses caused by different thermal intensity (Chapter 2). Furthermore, the responses after 4h (R4h) and 24h (R24h) stress stopped were investigated to evaluate the stress-recovery potentials of C. kiiensis (Chapter 3). In chapter 2, the results revealed increasing expressions of heat shock proteins (Hsps) for both HET and ET indicating enhancing stress resistances. In addition, DNA metabolisms were the major stress responses for HET rather for ET suggesting cellular damages caused by HET. Also, HET resulted energy deficiency in cells leading to obvious smaller survived individuals comparing to ET and CT. Comparing to HET, C. kiiensis was better acclimating to 24h ET with low effects on energy depletion and body growth. In chapter 3, our results demonstrated reversible transcriptomic responses for both HET and ET by recovering expression levels of Hsps at R24h and R4h respectively. Also, the effects of energy deficiency and cellular disorders caused by HET were recovered at R24h. This suggested recovered effects of unbalanced homeostasis and cellular damages. Moreover, the cellular repair systems remained essential processes for HET at R24h by activating ribosomal biogenesis. Although C. kiiensis could recover from 24h HET, the experimental individuals demonstrated trade-off effects at R24h with reduced body width, body length and antennal length. This confirmed that these stressed C. kiiensis sacrificed the body growth with devoting their energy for cellular repair and recovery processes. The results led to vulnerable status of individuals which suffered from HET by reducing successful rates of pupation. Overall, our findings suggested high acclimation capacity of C. kiiensis in response to both ET and HET by enhancing stress resistance and recovering from stress. However, our data also elucidated critical changes in C. kiiensis by increasing 1 ˚C from 39 ˚C to 40 ˚C leading to weaker individuals, i.e. smaller sizes, for pupation. This implied the vulnerable status of aquatic insects facing to future climate events of high intense heatwaves.