This study analyses the histological and cellular morphology of the testis and sperm development in the male Daphnia magna Straus 1820. Due to the rarity of males and predominately parthenogenetic lifecycle of Daphnia, there has been limited detailed information on males in contrast to the well-studied female. Using light and electron microscopy approaches, we describe the morphology of the testis during the progression from an immature to mature testis. The testis has an encasing muscular mesh sheath outside the basal lamina, beneath which is a thin somatic epithelial cell layer. Internal to the epithelium are the spermatogonial stem cells and subsequent syncytial clusters of the germ cells as they progress through spermatogenesis; spermatozoa occupy the entire testis in sexually mature D. magna. We describe the structure of developing and mature spermatozoa; mature spermatozoa are non-flagellated, ovoid in shape with plasmalemma filapodia and are encased in an extracellular capsule.

Daphnia spp. cycle between asexual and sexual reproduction depending on environmental conditions. Under favorable conditions, female offspring are produced, resulting in rapid population expansion; in less favorable conditions, males are produced, initiating the sexual portion of their lifestyle. Although mediated by environmental cues, sex determination is ultimately dependent on the expression of Doublesex genes, Dsx1 and Dsx2, in Daphnia. We exposed Daphnia magna to juvenile hormone III (JHIII), an insect hormone often mimicked by agricultural insecticides. Juvenile hormone (JH) and JH analogues disrupt the insect endocrine system, inhibiting reproduction; these chemicals pose an environmental threat to non-target organisms by leaching into surrounding waters. When Daphnia are exposed to JH or JH analogues, it causes a premature shift from asexual to sexual reproduction. We showed that Dsx1 is upregulated throughout vitellogenesis in female D. magna producing male offspring and Dsx2 is upregulated during late vitellogenesis, suggesting that both Dsx1 and Dsx2 contribute to male sex determination. In addition, Dsx2 expression increases during sexual maturation in male D. magna, suggesting that it also plays a role in spermatogenesis. In addition, exposure to JHIII decreased reproductive output, slowed growth and prematurely shifted D. magna reproductive strategy to the sexual portion of their lifecycle.

The symbiosis between cnidarians and dinoflagellates underpins the success of reef-building corals in otherwise nutrient-poor habitats. Alterations to symbiotic state can perturb metabolic homeostasis and thus alter the release of biogenic volatile organic compounds (BVOCs). While BVOCs can play important roles in metabolic regulation and signalling, how the symbiotic state affects BVOC output remains unexplored. We therefore characterised the suite of BVOCs that comprise the volatilome of the sea anemone Exaiptasia diaphana (‘Aiptasia’) when aposymbiotic and in symbiosis with either its native dinoflagellate symbiont Breviolum minutum or the non-native symbiont Durusdinium trenchii. In parallel, the bacterial community structure in these different symbiotic states was fully characterised to resolve the holobiont microbiome. Based on rRNA analyses, 147 unique amplicon sequence variants (ASVs) were observed across symbiotic states. Furthermore, the microbiomes were distinct across the different symbiotic states: bacteria in the family Vibrionaceae were the most abundant in aposymbiotic anemones; those in the family Crocinitomicaceae were the most abundant in anemones symbiotic with D. trenchii; and anemones symbiotic with B. minutum had the highest proportion of low-abundance ASVs. Across these different holobionts, 142 BVOCs were detected and classified into 17 groups based on their chemical structure, with BVOCs containing multiple functional groups being the most abundant. Isoprene was detected in higher abundance when anemones hosted their native symbiont, and dimethyl sulphide was detected in higher abundance in the volatilome of both Aiptasia-Symbiodiniaceae combinations relative to aposymbiotic anemones. The volatilomes of aposymbiotic anemones and anemones symbiotic with B. minutum were distinct, while the volatilome of anemones symbiotic with D. trenchii overlapped both of the others. Collectively, our results are consistent with previous reports that D. trenchii produces a metabolically sub-optimal symbiosis with Aiptasia, and add to our understanding of how symbiotic cnidarians, including corals, may respond to climate change should they acquire novel dinoflagellate partners.

The establishment and maintenance of the symbiosis between a cnidarian host and its dinoflagellate symbionts is central to the success of coral reefs. To explore the metabolite production underlying this symbiosis, we focused on a group of low weight secondary metabolites, biogenic volatile organic compounds (BVOCs). BVOCs are released from an organism or environment, and can be collected in the gas phase, allowing non-invasive analysis of an organism's metabolism (i.e. ‘volatilomics’). We characterised volatile profiles of the sea anemone Exaiptasia diaphana (‘Aiptasia’), a model system for cnidarian–dinoflagellate symbiosis, using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry. We compared volatile profiles between: 1) symbiotic anemones containing their native symbiont, Breviolum minutum; 2) aposymbiotic anemones; and 3) cultured isolates of B. minutum. Overall, 152 BVOCs were detected, and classified into 14 groups based on their chemical structure, the most numerous groups being alkanes and aromatic compounds. A total of 53 BVOCs were differentially abundant between aposymbiotic anemones and B. minutum cultures; 13 between aposymbiotic and symbiotic anemones; and 60 between symbiotic anemones and cultures of B. minutum. More BVOCs were differentially abundant between cultured and symbiotic dinoflagellates than between aposymbiotic and symbiotic anemones, suggesting that symbiosis may modify symbiont physiology more than host physiology. This is the first volatilome analysis of the Aiptasia model system and provides a foundation from which to explore how BVOC production is perturbed under environmental stress, and ultimately the role they play in this important symbiosis.