Adaptive evolution associated with dispensable gene loss is widespread in the animal kingdom. However, loss of genes essential for embryonic survival and the mechanisms that evolved to bypass negative selection and extinction are less well known. Here, we show that all Gadiformes, an ecologically dominant order of codfishes, lost the aqp1ab -type genes that are essential for the formation of hydrated pelagic eggs and embryonic survival of marine teleosts in oceanic environments. Despite such gene loss, codfishes produce some of the most highly hydrated pelagic eggs of all marine teleosts. To resolve this apparent paradox, we initially confirmed that the mechanisms of osmotic drive are conserved with other pelagic-egg-producing marine teleosts, and then conducted comparative transcript analyses of aquaporin water channels in distantly-related freshwater and marine teleosts. This uncovered Aqp4-type channels that have been chromosomally linked to the Aqp1-type channels in jawed vertebrate genomes for >450 million years as the primary candidates. Using immunolocalization, functional assays, and inhibition experiments with zebrafish and Atlantic cod as model organisms, we show that short isoforms of Aqp4 are required for egg ovulation in more ancestral lineages of freshwater teleosts and were recruited to the oocyte hydration pathway of codfishes. By contrast, the oldest and most modern lineages of marine teleosts producing pelagic eggs still utilize the aqp1ab genes for egg hydration. Our data thus reveal that codfishes survived essential gene loss by functionally co-opting freshwater ovulatory pathways to marine egg hydration.

In marine teleosts, the neuroendocrine regulation of aquaporin intracellular trafficking in osmoregulatory organs such as the intestinal rectum remains underexplored. By using paralog-specific antibodies, here we determined the subcellular localization of seven aquaporin and aquaglyceroporin paralogs in the rectal enterocytes of the marine teleost gilthead seabream (Sparus aurata) and investigated the neuropeptinergic effects of arginine vasopressin (Avp) and oxytocin (Oxt) on channel trafficking. The data reveal that Aqp1aa, -1ab2 and -3a are mainly localized in the cytoplasm and basolateral membranes of the enterocytes, while Aqp1ab1, -10bb and -14 are cytoplasmatic and also present in the apical plasma membrane microvilli. In contrast, Aqp0a localizes in the enterocyte subapical cytoplasm. Treatment of rectal explants with Avp in vitro increases the targeting of Aqp1ab1, -1ab2, -10bb and -14, but not of Aqp0a, -1aa and -3a, to the enterocyte apical microvilli, which involves protein kinase A (PKA)-mediated phosphorylation of the channel subdomains. In addition, we find that the differential interaction of the Aqp1ab-type paralogs with teleost-specific 14-3-3ζ-like binding proteins is associated with the trafficking of the channels to the apical microvilli membranes. Conversely, Oxt treatment does not affect the subcellular distribution of any of the investigated aquaporins, except Aqp14, which is internalized in response to the neuropeptide by a partially independent protein kinase C pathway. These data suggest that Avp-PKA signaling controls plasma membrane trafficking of several seabream rectal aquaporins, possibly to facilitate water and solute fluxes across the enterocyte membrane.

The insulin/insulin-like growth factor signaling (IIS) pathway plays a central role in several vital physiological processes in animals. The identification of three insulin receptor (InR) (BgInR1, BgInR2 and BgInR3) in the genome of the cockroach Blattella germanica (L.) prompted us to investigate into their respective contribution to the regulation of different events. Previous studies from our group revealed that BgInR2 is the paralog that retains most of the activity in reproduction-related functions. In the present study, we focused in elucidating the roles of the three InR of B. germanica in molting and analyzed their involvement in the hormonal regulation underlying the different phases of this process. To do this, we first quantified the expression of several genes, including ecdysteroidogenic genes and those of peptide hormones involved in molting. Our findings indicate that the ecdysis triggering hormone (ETH) is an ecdysteroid-responsive gene. Furthermore, we analyzed the phenotype produced by RNAi-triggered specific depletion of BgInR1, BgInR2 and BgInR3, and identified BgInR2 as the key factor in IIS signaling related to molting. BgInR2 depletion produced a delay and dysregulation in ecdysone production, which in turn led to reduced and delayed ETH expression. Finally, this culminated in defective ecdysis and impaired exuviae shedding. Overall, our results demonstrate that the IIS pathway, via BgInR2, promotes ecdysone synthesis in the prothoracic gland, which subsequently induces ETH production to initiate and coordinate ecdysis.

The intracellular unorthodox aquaporins, AQP11 and AQP12, remain the least characterized members of the aquaporin superfamily. Mammalian and teleost AQP12 channels have recently been established as polytransporters in native membranes. They are localized intracellularly in pancreatic acinar cells and considered to play roles in zymogen granule maturation and exocytosis. In contrast with the vertebrate channels, the permeability properties and physiological roles of invertebrate AQP12-like (Aqp12L) orthologs are completely unknown. Here, we report that the Aqp12L channel from the extremophile crustacean Artemia franciscana is specifically targeted to the membrane of the yolk platelets (YPs) when heterologously expressed in frog (Xenopus laevis) oocytes, as recently reported for the vertebrate orthologs. Using YPs isolated from A. franciscana Aqp12L-expressing frog oocytes, we further show that this channel can transport water, H2O2, ammonia, and urea but not glycerol. Immunostaining of the A. franciscana ovary using a specific Aqp12L antibody revealed that the in vivo localization of the channel also occurs in the YP membranes of vitellogenic oocytes. RNAi-mediated silencing of Aqp12L substantially reduces the offspring output of both ovoviviparous and oviparous females through the impairment of oocyte vitellogenesis (oocyte growth) and the premature activation of ovarian apoptotic and atretic pathways. These findings provide the first direct evidence demonstrating that invertebrate Aqp12L is a functional polytransporter with an essential role in crustacean oogenesis.