Molecular mechanisms underpinning buoyancy control in the aquatic larvae of Chaoborus evolved from tracheal fluid clearing functionality

Aquatic larvae belonging to the midge genus Chaoborus are the only truly pelagic insects, possessing the ability to regulate their buoyancy and position in the water column. They achieve this buoyancy control using two pairs of closed, air-filled sacs which are derived from the longitudinal trunks of their tracheal system. Previous work has revealed that the volume of these air-sacs is controlled using a unique mechanochemical system, where bands of the protein resilin alternate with bands of tracheal cuticle in the air-sac wall to produce a composite material which expands and contracts when exposed to changes in pH. While a simple epithelium enveloping each air-sac is known to control the pH of the resilin bands, the molecular mechanisms underpinning its ability to regulate pH, and thus control the air-sac’s volume, are unknown. To reveal these mechanisms, and how this functionality evolved, we compare the transcriptomes of air-sacs from Chaoborus trivitattus larvae with tracheal tissue from the aquatic larvae of two other genera within the Chaoboridae: Eucorethra underwoodi whose larvae possess an unmodified tracheal system which they use to breathe air using a posterior respiratory siphon, and Mochlonyx cinctipes whose larvae possess a tracheal system with dilated anterior and posterior sac-like regions, but which remains open through a posterior siphon. We found that Chaoborus air-sac epithelia show strong expression of ion channels related to pH regulation relative to Eucorethra tracheal tissue, including orthologs of NHA1, Nhe2, Ae2, and pHCl-2, as well as high levels of transcript abundance of the aquaporin water channel, Drip. This suggests that the tracheal epithelium of the ancestral chaoborid possesses all the functionality required to control pH and deal with the flux of water associated with the swelling and contraction of the resilin as part of the ancestral liquid-clearing function of the tracheal epithelium, setting the stage for the evolution of a novel buoyancy control mechanism.

属于幽蚊属（Chaoborus）的水生幼虫是目前已知唯一真正适配水层浮游生活的昆虫，它们可自主调控自身在水柱中的浮力与空间位置。这类幼虫借助两对源自气管系统纵干的封闭充气囊实现浮力调控。既往研究揭示，这些充气囊的体积受一套独特的机械化学系统管控：充气囊壁上，节肢弹性蛋白（resilin）条带与气管角质层（tracheal cuticle）条带交替排布，形成一种复合材料，可随pH值变化发生伸缩形变。虽然已知包裹每个充气囊的单层上皮细胞可调控节肢弹性蛋白条带所处的pH环境，但支撑其调节pH、进而控制充气囊体积的分子机制仍未阐明。为揭示该机制及其功能演化历程，我们将三斑幽蚊（Chaoborus trivitattus）幼虫的充气囊转录组（transcriptomes），与幽蚊科另外两个属的水生幼虫气管组织进行比对分析：其一为安德伍德真幽蚊（Eucorethra underwoodi），其幼虫拥有未经过修饰的气管系统，通过后端呼吸虹吸管呼吸空气；其二为带纹莫氏幽蚊（Mochlonyx cinctipes），其幼虫的气管系统具有扩张的前、后囊状区域，但仍通过后端虹吸管保持开放状态。研究发现，相较于安德伍德真幽蚊的气管组织，三斑幽蚊充气囊上皮细胞高表达与pH调控相关的离子通道，包括NHA1、Nhe2、Ae2与pHCl-2的直系同源基因（orthologs），同时水通道蛋白（aquaporin）Drip的转录本丰度也显著升高。该结果表明，幽蚊科祖先的气管上皮细胞已具备调控pH、以及应对节肢弹性蛋白伸缩伴随的水分流动所需的全部功能，这是气管上皮祖先液体清除功能的一部分，为新型浮力调控机制的演化奠定了基础。