體腔真菌對白線斑蚊病媒能力之影響

Abstract

蚊蟲傳播多種蚊媒疾病，對人類的健康造成很大的威脅，過度依賴傳統的化學性殺蟲劑會造成蚊蟲抗藥性並對環境帶來負面影響，因此蚊蟲的綜合防治策略逐漸受到重視。專性寄生的體腔真菌，主要藉由在橈足類和蚊蟲族群間完成生活史，並常有高達 90% 以上的感染率，且會在宿主蚊蟲族群內發生流行病，使其具有防治蚊蟲的潛能。已知全球體腔真菌有 84 種，分類上主要依賴形態分類，國家生物技術資訊中心上發表的體腔真菌序列計有四種，臺灣尚缺整合形態與分子證據的研究報告。本研究藉由野外蚊蟲幼蟲的田野調查來研究體腔真菌，並通過掃描式電子顯微鏡和分子序列對體腔真菌進行形態和分子鑑定；透過實驗室感染白線斑蚊幼蟲以評估對體腔真菌的感受性，以及評估其防治蚊幼蟲的潛能，並藉由即時定量聚合酶連鎖反應去定量白線斑蚊幼蟲感染體腔真菌後的免疫調節機制。最後對感染體腔真菌的雌蟲餵食登革病毒，以即時定量聚合酶連鎖反應定量蚊蟲體內各個器官感染的病毒效價，以探討感染體腔真菌的成蟲，在體內與登革病毒的交互作用，及其感染體腔真菌後的病媒能力。田野調查於臺灣地區中北部的11個孳生源中，採集到被體腔真菌感染的白線斑蚊幼蟲，並以 18S SSU rDNA 序列和形態特徵鑑定出 Coelomomyces macleayae， C. stegomyiae var. stegomyiae 和 C. stegomyiae var. chapmani，和國家生物技術資訊中心上的 C. stegomyiae 序列相似度分別為88.77、95.58 和 99.59%，在實驗室中以白線斑蚊和異足猛水蚤建立體腔真菌族群 (C. macleayae)。幼蟲對於體腔真菌的感受性，在 102.69 RS/ml 濃度下的體腔真菌 (C. macleayae)，一齡幼蟲感染率 (90.67±4.16%，136/150) 高於二齡幼蟲 (60.67±5.77%，91/150)，三齡幼蟲在 103.12 RS/ml 濃度下，感染率為 46.67±6.67%。體腔真菌 (C. macleayae) 對於蚊蟲免疫基因的調控，Rel1 基因的表現量在感染後第八天顯著上升， Rel2 基因的表現量則是在感染後第四天顯著上升，顯示體腔真菌的感染會引起蚊蟲免疫反應的 Toll、IMD 路徑。感染體腔真菌 (C. macleayae) 的成蟲有壽命顯著縮短的現象，且使其短於登革病毒在白線斑蚊的體外潛伏期，並以這種縮短壽命的方式來降低白線斑蚊的病媒能力。

Mosquitoes are of great threat to human health because they transmit vector-borne diseases. Integrated vector management for mosquito control has gained interest rather than using chemical insecticides. Obligatory parasitic fungi, species of Coelomomyces (Blastocladiomycetes: Blastocladiales), have potential roles for mosquito control based on the advantages of high prevalence (often higher than 90%) and lethal effects. Four partial sequence data of Coelomomyces have been identified, however, data in Taiwan has not yet been available. Aedes albopictus were collected from northern Taiwan, and Coelomomyces were identified by both molecular method and scanning electron microscope. The potential of Coelomomyces on mosquito control was evaluated based on the survival rate of larvae. The modulation of antifungal immune responses in mosquito larvae to Coelomomyces infection was also estimated by RT-qPCR, including Rel1, Rel2, STAT, and TEP1. Interactions between Coelomomyces and dengue virus (DENV) in female adults were investigated by oral feeding of virus-infected blood. Vector competence and interactions between DENV and Coelomomyces in mosquito tissues were quantified by RT-qPCR. Ae. albopictus and Canthocamptus cf. kunzi were co-cultured in the laboratory to maintain Coelomomyces in vivo. Sequences of 18S SSU rDNA from three samples shows 88.77, 95.58, and 99.59% similarity to C. stegomyiae (Accession number: AF322406.1). The molecular and morphological data indicate that one novel strain of C. macleayae, and two strains of C. stegomyiae var. stegomyiae and C. stegomyiae var. chapmani were isolated. The infection rate of C. macleayae in the 1st instar larvae (90.67±4.16%, 136/150) was higher than the 2nd instar larvae (60.67±5.77%, 91/150) after incubating with 102.69 resting sporangia/ml solution for 2 days, and the mortality rates were 72.96±9.67% and 47.39±6.20%, respectively. In the 3rd instar larvae, the infection rate was 46.67±6.67%, and the mortality rate was 37.35±5.52% in 103.12 resting sporangia/ml solution (N=90). The expressions of Rel1 at 8d post-infection and Rel2 at 4d post-infection in the Ae. albopictus 3rd larvae infected by C. macleayae were significantly upregulated. It showed that the infection of Coelomomyces would activate the Toll and IMD pathway in the immune system of mosquito larvae. The life span of mosquitoes infected by C. macleayae significantly shortened to less than the EIP of dengue virus in Ae. albopictus and Coelomomyces may decrease the vectorial capacity through a shortening life span.