Biphasic Life Cycle Research Articles

The oldest tadpole reveals evolutionary stability of the anuran life cycle.

Anurans are characterized by a biphasic life cycle, with an aquatic larval (tadpole) stage followed by an adult (frog) stage, both connected through the metamorphic period in which drastic morphological and physiological changes occur1. Extant tadpoles exhibit great morphological diversity and ecological relevance2, but their absence in the pre-Cretaceous fossil record (older than 145 million years) makes their origins and early evolution enigmatic. This contrasts with the postmetamorphic anuran fossil record that dates back to theEarly Jurassic and with closely related species in the Late Triassic (around 217-213 million years ago (Ma))3. Here we report a late-stage tadpole of the stem-anuran Notobatrachus degiustoi from the Middle Jurassic of Patagonia (around 168-161 Ma). This finding has dual importance because it represents the oldest-known tadpole and, to our knowledge, the first stem-anuran larva. Its exquisite preservation, including soft tissues, shows features associated with the filter-feeding mechanism characteristic of extant tadpoles4,5. Notably, both N. degiustoi tadpole and adult reached a large size, demonstrating that tadpole gigantism occurred among stem-anurans. This new discovery reveals that a biphasic life cycle, with filter-feeding tadpoles inhabiting aquatic ephemeral environments, was already present in the early evolutionary history of stem-anurans and has remained stable for at least 161 million years.

Evaluating STING Agonist Impact on Kaposi’s Sarcoma-Associated Virus-related Cancers

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an etiological agent of multiple malignancies associated with compromised immunity, such as Kaposi’s sarcoma (KS) and Primary Effusion Lymphoma (PEL). KSHV infection is lifelong with no available treatment or vaccines and poses a high risk to immunocompromised individuals. The virus is transmitted via the oral and sexual route, and has a biphasic life cycle with a short lytic replication cycle followed by long-term latency, with the expression of few genes making detection difficult. Reactivation from latency results in a cascade of viral gene expression and pathogenesis. Critical to controlling KSHV infection is the cGAS-STING DNA-sensing pathway (STING), known to activate the type I interferon response and induce cancer cell death or growth inhibition. STING exhibits both anti-viral and anti-tumor responses, showing promise as a protein of interest to study for the development of a therapeutic treatment. By examining the in vitro effectiveness of STING agonists in inhibiting KSHV oncogenesis in PEL, the study aimed to provide the foundation for a potential novel therapeutic approach. Six patient-derived PEL cell lines were treated with the STING agonist diaminobenzamidazole (diABZI) in a dose-dependent manner. Four of the PEL cell lines exhibited decreased cell growth, viability, and colony formation, while two PEL cell lines were resistant to treatment, correlating with the presence or absence of STING expression. RNA-sequencing was performed and discovered upregulation of multiple interferon-stimulated genes (ISG) in responsive cell lines. Further investigations will explore STING agonists’ inhibition of PEL oncogenesis in vitro via colony formation and in vivo using a PEL xenograft model with NSG mice. Successful completion of this project will offer insight into potential clinical treatments for targeting KSHV infection and reactivation.

Contrasting the development of larval and adult body plans during the evolution of biphasic lifecycles in sea urchins.

Biphasic lifecycles are widespread among animals, but little is known about how the developmental transition between larvae and adults is regulated. Sea urchins are a unique system for studying this phenomenon because of the stark differences between their bilateral larval and pentaradial adult body plans. Here, we use single-cell RNA sequencing to analyze the development of Heliocidaris erythrogramma (He), a sea urchin species with an accelerated, non-feeding mode of larval development. The sequencing time course extends from embryogenesis to roughly a day before the onset of metamorphosis in He larvae, which is a period that has not been covered by previous datasets. We find that the non-feeding developmental strategy of He is associated with several changes in the specification of larval cell types compared to sea urchins with feeding larvae, such as the loss of a larva-specific skeletal cell population. Furthermore, the development of the larval and adult body plans in sea urchins may utilize largely different sets of regulatory genes. These findings lay the groundwork for extending existing developmental gene regulatory networks to cover additional stages of biphasic lifecycles.

Developmental histology of the liver in the Balkan crested newt Triturus ivanbureschi (Caudata: Salamandridae)

The liver, a crucial organ for metabolic processes, has a generally uniform histological structure across amphibian taxa. However, ontogenetic changes, particularly those related to biphasic life cycle and metamorphosis, are less documented. Here, we explored and described the liver histology of an emerging model organism, the Balkan crested newt (Triturus ivanbureschi) at three ontogenetic stages: larval, juvenile (just after metamorphosis) and adult. At the larval stage, the liver is characterized by hepatocytes containing large lipid droplets, poorly developed basement membranes in the blood vessels, and a lack of melanin-rich macrophage centers. Juveniles show transitional characteristics between larvae and adults. Lipid droplets in hepatocytes are abundant, but also, at the juvenile stage the well-developed basement membrane of blood vessels and melanomacrophages are present, as in adults. The presence of lipid droplets in hepatocytes during larval and juvenile stages suggests the liver's role in fat storage and energy provision during development and growth. Melanomacrophages, which synthesize melanin, perform phagocytosis, and neutralize free radicals, have been found in juveniles (after metamorphosis) and increase with age. The biphasic life cycle and liver histology transition in Triturus newts provide an insight in changes in liver histology and make them a suitable model for studying fat deposition regulation and the evolution of the immune system in terrestrial vertebrates.

Does habitat modification impact morphology, performance, and inflammatory responses in an amphibian with limited dispersal capacity (Lisssotriton helveticus)?

The environment of an organism exerts selective pressures that affect mobility, feeding, reproduction as well as predator-prey and conspecific interactions. Land use changes induced by human activities modify these selective pressures and may result in the adaptation of organisms. Amphibians are ectotherms that typically show a biphasic life cycle with an aquatic and terrestrial phase, which makes them particularly sensitive to environmental change. We studied the impact of habitat modifications on palmate newt populations in the Ile de France region across four types of habitats: urban, mixed, agricultural, and natural with at least two replicates for each habitat type. We measured the morphology of newts using callipers, quantified maximal running and swimming speed and acceleration using high-speed video recordings, and quantified the swelling of the hind limb linked to an inflammatory reaction. Our results show that in urban habitats, newts are larger and heavier and have a better body condition. Females, moreover, have a larger head in natural habitats, possibly due to diet specialisation of females during the breeding season. In mixed and agricultural habitats, newts have longer limbs and show a tendency to run faster, possibly associated with the selective pressures on movement in mixed habitats. Differences in inflammatory responses were observed between sexes but not habitat types. Overall, our results show differences in morphology and trends for differences in performance in newts living in different habitats suggesting that animals are adapting to human-induced changes in their environment.

The nucleic acid binding protein SFPQ represses EBV lytic reactivation by promoting histone H1 expression.

Epstein-Barr virus (EBV) uses a biphasic lifecycle of latency and lytic reactivation to infect >95% of adults worldwide. Despite its central role in EBV persistence and oncogenesis, much remains unknown about how EBV latency is maintained. We used a human genome-wide CRISPR/Cas9 screen to identify that the nuclear protein SFPQ was critical for latency. SFPQ supported expression of linker histone H1, which stabilizes nucleosomes and regulates nuclear architecture, but has not been previously implicated in EBV gene regulation. H1 occupied latent EBV genomes, including the immediate early gene BZLF1 promoter. Upon reactivation, SFPQ was sequestered into sub-nuclear puncta, and EBV genomic H1 occupancy diminished. Enforced H1 expression blocked EBV reactivation upon SFPQ knockout, confirming it as necessary downstream of SFPQ. SFPQ knockout triggered reactivation of EBV in B and epithelial cells, as well as of Kaposi's sarcoma-associated herpesvirusin B cells, suggesting a conserved gamma-herpesvirus role. These findings highlight SFPQ as a major regulator of H1 expression and EBV latency.

Amphibians at risk: Effects of climate change in the southwestern North American drylands

Climate change can affect species' geographical range in several ways, leading to species' geographical range expansion and contraction. Amphibians are the most threatened vertebrate class, and deterioration is due, in part, to the loss of suitable habitat. This taxonomic group is especially sensitive because of their biphasic life cycles (aquatic and terrestrial) and microhabitat requirements. Herein, we propose a combined approach, which uses biological and life history information to understand species' sensitivity to climate change. The goals of our study were to (1) identify the contribution of environmental variables to the broad-scale distribution of amphibians in drylands of the southwestern United States and Mexico, (2) describe how the habitat suitability of amphibians can potentially respond to a range of climate change scenarios across drylands, and (3) identify amphibian traits related to increased vulnerability and adaptation to the specific pressures and mechanisms of climate change projected for the dryland areas. Results show that climate strongly influences the geographic distribution of amphibian diversity in drylands and that most amphibians are expected to lose habitat in the upcoming decades. This study also shows that areas with the greatest loss of high habitat suitability are related to temperature increases and precipitation reductions. Our results build a framework that will significantly improve the current state of knowledge on the drylands' biogeography while providing comprehensive information and analyses for decision-makers to conserve and protect endangered and threatened amphibian species and their habitats.

Diversity and Evolution of Frog Visual Opsins: Spectral Tuning and Adaptation to Distinct Light Environments.

Visual systems adapt to different light environments through several avenues including optical changes to the eye and neurological changes in how light signals are processed and interpreted. Spectral sensitivity can evolve via changes to visual pigments housed in the retinal photoreceptors through gene duplication and loss, differential and coexpression, and sequence evolution. Frogs provide an excellent, yet understudied, system for visual evolution research due to their diversity of ecologies (including biphasic aquatic-terrestrial life cycles) that we hypothesize imposed different selective pressures leading to adaptive evolution of the visual system, notably the opsins that encode the protein component of the visual pigments responsible for the first step in visual perception. Here, we analyze the diversity and evolution of visual opsin genes from 93 new eye transcriptomes plus published data for a combined dataset spanning 122 frog species and 34 families. We find that most species express the four visual opsins previously identified in frogs but show evidence for gene loss in two lineages. Further, we present evidence of positive selection in three opsins and shifts in selective pressures associated with differences in habitat and life history, but not activity pattern. We identify substantial novel variation in the visual opsins and, using microspectrophotometry, find highly variable spectral sensitivities, expanding known ranges for all frog visual pigments. Mutations at spectral-tuning sites only partially account for this variation, suggesting that frogs have used tuning pathways that are unique among vertebrates. These results support the hypothesis of adaptive evolution in photoreceptor physiology across the frog tree of life in response to varying environmental and ecological factors and further our growing understanding of vertebrate visual evolution.

Opsin expression varies across larval development and taxa in pteriomorphian bivalves.

Many marine organisms have a biphasic life cycle that transitions between a swimming larva with a more sedentary adult form. At the end of the first phase, larvae must identify suitable sites to settle and undergo a dramatic morphological change. Environmental factors, including photic and chemical cues, appear to influence settlement, but the sensory receptors involved are largely unknown. We targeted the protein receptor, opsin, which belongs to large superfamily of transmembrane receptors that detects environmental stimuli, hormones, and neurotransmitters. While opsins are well-known for light-sensing, including vision, a growing number of studies have demonstrated light-independent functions. We therefore examined opsin expression in the Pteriomorphia, a large, diverse clade of marine bivalves, that includes commercially important species, such as oysters, mussels, and scallops. Genomic annotations combined with phylogenetic analysis show great variation of opsin abundance among pteriomorphian bivalves, including surprisingly high genomic abundance in many species that are eyeless as adults, such as mussels. Therefore, we investigated the diversity of opsin expression from the perspective of larval development. We collected opsin gene expression in four families of Pteriomorphia, across three distinct larval stages, i.e., trochophore, veliger, and pediveliger, and compared those to adult tissues. We found larvae express all opsin types in these bivalves, but opsin expression patterns are largely species-specific across development. Few opsins are expressed in the adult mantle, but many are highly expressed in adult eyes. Intriguingly, opsin genes such as retinochrome, xenopsins, and Go-opsins have higher levels of expression in the later larval stages when substrates for settlement are being tested, such as the pediveliger. Investigating opsin gene expression during larval development provides crucial insights into their intricate interactions with the surroundings, which may shed light on how opsin receptors of these organisms respond to various environmental cues that play a pivotal role in their settlement process.

Listeria monocytogenes utilizes glutathione and limited inorganic sulfur compounds as sources of essential cysteine.

Listeria monocytogenes (Lm) is a Gram-positive facultative intracellular pathogen that leads a biphasic lifecycle, transitioning its metabolism and selectively inducing virulence genes when it encounters mammalian hosts. Virulence gene expression is controlled by the master virulence regulator PrfA, which is allosterically activated by the host- and bacterially derived glutathione (GSH). The amino acid cysteine is the rate-limiting substrate for GSH synthesis in bacteria and is essential for bacterial growth. Unlike many bacteria, Lm is auxotrophic for cysteine and must import exogenous cysteine for growth and virulence. GSH is enriched in the host cytoplasm, and previous work suggests that Lm utilizes exogenous GSH for PrfA activation. Despite these observations, the import mechanism(s) for GSH remains elusive. Analysis of known GSH importers predicted a homologous importer in Lm comprised of the Ctp ABC transporter and the OppDF ATPases of the Opp oligopeptide importer. Here, we demonstrated that the Ctp complex is a high-affinity GSH/GSSG importer that is required for Lm growth at physiologically relevant concentrations. Furthermore, we demonstrated that OppDF is required for GSH/GSSG import in an Opp-independent manner. These data support a model where Ctp and OppDF form a unique complex for GSH/GSSG import that supports growth and pathogenesis. In addition, we show that Lm utilizes the inorganic sulfur sources thiosulfate and H2S for growth in a CysK-dependent manner in the absence of other cysteine sources. These findings suggest a pathoadaptive role for partial cysteine auxotrophy in Lm, where locally high GSH/GSSG or inorganic sulfur concentrations may signal arrival to distinct host niches.

Tadpoles in lotic waters, habitat specialization, and human population density lead tree frogs (Hylinae) to higher extinction risk

Analyzing patterns of vulnerability to extinction across clades and traits that make species more prone to extinction are crucial steps towards prioritization of conservation efforts. Here we identified genera more vulnerable to extinction than expected by chance within the anuran subfamily Hylinae. We also investigated a broad range of intrinsic and extrinsic factors and their interactions as predictors of a proxy for extinction risk (IUCN Red List status). By conducting simulation analyses, we found that binary threat status is not randomly distributed across Hylinae genera, with Charadrahyla, Hyloscirtus, Isthmohyla, Plectrohyla, and Sarcohyla containing more threatened species than expected by chance. By using model selection and average approaches, we found strong support for increased likelihood of threatened status as habitat diversity decreases and human population density increases, and for species with larvae inhabiting lotic waters to be more threatened than those with larvae inhabiting lentic waters. Overall, our results were robust to different treatments of Data Deficient species (threatened or non-threatened). However, results were affected by the inclusion of geographic range size as another predictor, with habitat diversity becoming a non-significant predictor of threatened status. Our results highlight the importance of simultaneously analyzing both intrinsic and extrinsic predictors in correlative studies of extinction risk. Additionally, they underscore the need for conservation research and programs focused on both larvae and adults of organisms with biphasic life cycles and the establishment and evaluation of effectiveness of natural refuges far from areas under high human pressures, such as privately and community protected areas.

Relationships of primary productivity with anuran abundance, richness, and community composition in tropical streams.

The relationship between primary productivity and diversity has been demonstrated across taxa and spatial scales, but for organisms with biphasic life cycles, little research has examined whether productivity of larval and adult environments influence each life stage independently, or whether productivity of one life stage's environment outweighs the influence of the other. Experimental work demonstrates that tadpoles of stream-breeding anurans can exhibit a top-down influence on aquatic primary productivity (APP), but few studies have sought evidence of a bottom-up influence of primary productivity on anuran abundance, species richness and community composition, as seen in other organisms. We examined aquatic and terrestrial primary productivity in two forest types in Borneo, along with amphibian abundance, species richness, and community composition at larval and adult stages, to determine whether there is evidence for a bottom-up influence of APP on tadpole abundance and species richness across streams, and the relative importance of aquatic and terrestrial primary productivity on larval and adult phases of anurans. We predicted that adult richness, abundance, and community composition would be influenced by terrestrial primary productivity, but that tadpole richness, abundance, and community composition would be influenced by APP. Contrary to expectations, we did not find evidence that primary productivity, or variation thereof, predicts anuran richness at larval or adult stages. Further, no measure of primary productivity or its variation was a significant predictor of adult abundance, or of adult or tadpole community composition. For tadpoles, we found that in areas with low terrestrial primary productivity, abundance was positively related to APP, but in areas with high terrestrial primary productivity, abundance was negatively related to APP, suggesting a bottom-up influence of primary productivity on abundance in secondary forest, and a top-down influence of tadpoles on primary productivity in primary forest. Additional data are needed to better understand the ecological interactions between terrestrial primary productivity, aquatic primary productivity, and tadpole abundance.

First ultrastructural description of an apomictic opsiblastic egg in freshwater Gastrotricha.

Freshwater gastrotrichs have a biphasic lifecycle that reputedly involves the production of three types of eggs: apomictic and fast hatching (tachyblastic ova), apomictic and delayed hatching (opsiblastic ova), and plaque-bearing eggs (potentially derived from mixis). While some details of oogenesis and eggshell structure are known for tachyblastic ova, there are few details on other egg types. Here, we provide the first ultrastructural description of the oviposited opsiblastic eggs of the freshwater gastrotrich, Lepidodermella squamata. Scanning electron microscopy revealed the eggshell surface to be ornamented with long flattened pillar-like structures centered on polygonal plates that are pitted along their periphery. Transmission electron microscopy showed the pits to lead to a vast labyrinth of tubular spaces and larger cavities throughout the thick apical layer of the shell. The basal layer of the shell is amorphous and connected to a network of fine fibers that traverse an extra-oocyte space and forms a protective sheet around the uncleaved oocyte. The uncleaved oocyte has a dense layer of peripheral ooplasm surrounding a core of organelles including mitochondria, membrane-bound secretion granules, endoplasmic reticulum, and a single nucleus in a granular, ribosome-rich cytoplasm. Secretion granules are the most abundant organelles and presumably contain lipid-rich yolk that will be used as energy for delayed cleavage, thus functioning in temporal dispersal. These data are compared to the fine structure of invertebrate resting eggs across the phylogenetic spectrum to determine the novelty of opsiblastic egg structure in L. squamata.

Global shortfalls of knowledge on anuran tadpoles

Despite the amount of data on different aspects of biodiversity, such as species distributions, taxonomy, or phylogenetics, there are still significant gaps and biases in the available information. This is particularly true for life history traits, with fragmentary data for most taxa, especially those with complex life cycles. Anurans (frogs and toads) show larval (premetamorphic) stages that are in general radically decoupled from adult forms in most biological aspects. Our understanding of this group is highly uneven, as the main wide-scope investigations focus on adult specimens and larval stages remain unknown for a significant part of the anuran tree. The main purpose of this work was to estimate the extent of knowledge gaps regarding the diversity of tadpoles, interpret their biological and geographical patterns, and discuss possible explanations and implications for other large-scale analyses. Our findings show that more than half of the anuran species described to date still lack information on their embryonic/larval stages. Furthermore, knowledge varies among taxonomic groups, larval ecomorphological guilds, and world ecoregions. Description percentages generally decrease in lineages with a higher proportion of species known or suspected to have endotrophic development. Also, geographic areas with the highest levels of ignorance in larval biology (Tropical Andes and New Guinea) coincide with the highest diversity of endotrophic guilds. Among exotrophic larvae, generalized lentic-lotic tadpoles have the widest distribution and levels of knowledge, whereas specialized lotic, fossorial, and terrestrial forms are more taxonomically and geographically restricted. Further large-scale analyses on tadpole biology are crucial for their impact in varied scientific disciplines including anuran conservation. At a conceptual level, the discussion of the anuran biphasic life cycle is pertinent in the context of shortfalls of biodiversity knowledge and their interrelationships.

Following the footsteps of Burmeister's leaf frog (Phyllomedusa burmeisteri) in the Atlantic forest of Brazil

Amphibians are organisms which mainly have a biphasic life cycle. When at the larval stage, their habitat is generally aquatic, and when adults, they become terrestrial. Pond-breeding amphibians are sensitive to some disturbances in their environment which lead to the decline of a population. The interactions between the species and their environment are performed through movement. Movement ecology combines and explains the movement data of organisms with biotic and abiotic factors and because of this, knowing the movement of these creatures is of great ecological importance. We used the spool-and-line methodology in individuals of the treefrog Phyllomedusa burmeisteri, at the Reserva Ecologica Michelin, located in the southern region of Bahia in Brazil to study their movement patterns in different environments inside and outside of the forest. We monitored 19 P. burmeisteri individuals that presented a mean total movement distance of 2160.76 cm (S.D. 1152.42). We found no significant difference in the trajectories of individuals in forested and open areas, as well as a positive relationship between the distances individuals moved and their weight only in forested areas. no relationship between distance and weight of these individuals. We observed that individuals followed non-linear paths and the number of steps from one place to another varied among individuals. Our movement ecology data allows us to answer questions about short-term movement patterns of Phyllomedusa burmeisteri. This is the first step to start understanding the spatial cognition of treefrogs from the Atlantic Forest and to fill gaps about life habits of these frogs. Information on the movement patterns of a species, as well as its home range can help to create conservation strategies, regarding the creation or delimitation of protected areas, for example.

The biphasic life cycle of the non-spinose planktonic foraminifera is characterised by an aberrant coiling signature

The complete lifecycle of the planktonic foraminifera has continued to remain obscure. Benthic foraminifera exhibit an obligate alternation of generations between haploid gamonts and diploid agamonts, yet despite years of observation, the fusion of gametes to form a zygote, gamont or agamont has never been observed in the planktonic groups. Recent evidence from Neogloboquadrina pachyderma culture confirms that they do have a biphasic reproductive cycle and the agamonts may exhibit dimorphism in coiling direction. In this study we examine the morphology of normal and aberrant coiling tests of both N. pachyderma and N. incompta from three Arctic and North Atlantic water masses, to explore whether aberrant coiling is an indicator of a biphasic life cycle in the non-spinose planktonic foraminifera group as a whole. Using a Nano-Computed Tomography scanning approach, we morphologically compared the proloculus and growth trajectories of aberrant and normal coiling tests. Aberrant coiling tests were found to have a slightly larger proloculus and test size, consistent with a left coiling N. pachyderma agamont in the literature. This degree of dimorphism is insufficient to discriminate agamonts in the wild population and coiling direction remains their only distinguishable feature without further morphological investigation. The constant low-level aberrant coiling signature in a range of non-spinose planktonic foraminiferal genera implies that a biphasic life cycle is the principal mode of reproduction in the non-spinose planktonic group.

Ticks and Chlamydia-Related Bacteria in Swiss Zoological Gardens Compared to in Contiguous and Distant Control Areas.

Ticks are vectors of numerous agents of medical importance and may be infected by various Chlamydia-related bacteria, such as members of Parachlamydiaceae and Rhabdochlamydiaceae families, which are sharing the same biphasic life cycle with the pathogenic Chlamydia. However, the veterinary importance of ticks and of their internalized pathogens remains poorly studied. Thus, we wondered (i) whether the prevalence of ticks was higher in zoological gardens than in control areas with similar altitude, vegetation, humidity and temperature, and (ii) whether the presence of Chlamydia-related bacteria in ticks may vary according to the environment in which the ticks are collected. A total of 212 Ixodes ricinus ticks were collected, and all were tested for the presence of DNA from any member of the Chlamydiae phylum using a pan-Chlamydiae quantitative PCR (qPCR). We observed a higher prevalence of ticks outside animal enclosures in both zoos, compared to in enclosures. Tick prevalence was also higher outside zoos, compared to in enclosures. With 30% (3/10) of infected ticks, the zoological gardens presented a prevalence of infected ticks that was higher than that in contiguous areas (13.15%, 10/76), and higher than the control distant areas (8.65%, 9/104). In conclusion, zoological gardens in Switzerland appear to contain fewer ticks than areas outside zoological gardens. However, ticks from zoos more often contain Chlamydia-like organisms than ticks from contiguous or distant control areas.

RNA Editing-Dependent and -Independent Roles of Adenosine Deaminases Acting on RNA Proteins in Herpesvirus Infection-Hints on Another Layer of Complexity.

The Adenosine Deaminases Acting on RNA (ADAR) catalyze the posttranscriptional deamination of adenosine residues to inosine in double-stranded RNAs (dsRNAs, A-to-I editing), preventing the overactivation of dsRNA sensor molecules and interferons. RNA editing is the cornerstone of innate immunity that distinguishes between self and non-self (virus), and it is essential for normal regulation of cellular homeostasis. Although much is already known about the role of ADAR proteins in RNA virus infection, the role of ADAR proteins in herpesvirus infection remains largely unexplored. In this review, we provide several lines of evidence from studies of different herpesviruses for another level of complexity in regulating the already intricate biphasic life cycle of herpesviruses.

Using CRISPR/Cas9 to identify genes required for mechanosensory neuron development and function.

Tunicates are marine, non-vertebrate chordates that comprise the sister group to the vertebrates. Most tunicates have a biphasic lifecycle that alternates between a swimming larva and a sessile adult. Recent advances have shed light on the neural basis for the tunicate larva's ability to sense a proper substrate for settlement and initiate metamorphosis. Work in the highly tractable laboratory model tunicate Ciona robusta suggests that sensory neurons embedded in the anterior papillae transduce mechanosensory stimuli to trigger larval tail retraction and initiate the process of metamorphosis. Here, we take advantage of the low-cost and simplicity of Ciona by using tissue-specific CRISPR/Cas9-mediated mutagenesis to screen for genes potentially involved in mechanosensation and metamorphosis, in the context of an undergraduate 'capstone' research course. This small screen revealed at least one gene, Vamp1/2/3, which appears crucial for the ability of the papillae to trigger metamorphosis. We also provide step-by-step protocols and tutorials associated with this course, in the hope that it might be replicated in similar CRISPR-based laboratory courses wherever Ciona are available.

Projecting kelp (Ecklonia radiata) gametophyte thermal adaptation and persistence under climate change.

Kelp forests underpin temperate marine ecosystems but are declining due to ocean warming, causing loss of associated ecosystem services. Projections suggest significant future decline but often only consider the persistence of adult sporophytes. Kelps have a biphasic life cycle, and the haploid gametophyte can be more thermally tolerant than the sporophyte. Therefore, projections may be altered when considering the thermal tolerance of gametophytes. We undertook thermal tolerance experiments to quantify the effect of temperature on gametophyte survival, relative growth rate (RGR) and sex ratio for three genetically distinct populations of Ecklonia radiata gametophytes from comparatively high, mid- and low latitudes (43°, 33° and 30°S). We then used these data to project the likely consequences of climate-induced thermal change on gametophyte persistence and performance across its eastern Australian range, using generalized additive and linear models. All populations were adapted to local temperatures and their thermal maximum was 2-3 °C above current maximum in situ temperatures. The lowest latitude population was most thermally tolerant (~70 % survival up to 27 °C), while survival and RGR decreased beyond 25.5 and 20.5 °C for the mid- and low-latitude populations, respectively. Sex ratios were skewed towards females with increased temperature in the low- and high-latitude populations. Spatially explicit model projections under future ocean warming (2050-centred) revealed a minimal decline in survival (0-30 %) across populations, relative to present-day predictions. RGRs were also projected to decline minimally (0-2 % d-1). Our results contrast with projections for the sporophyte stage of E. radiata, which suggest a 257-km range contraction concurrent with loss of the low-latitude population by 2100. Thermal adaptation in E. radiata gametophytes suggests this life stage is likely resilient to future ocean warming and is unlikely to be a bottleneck for the future persistence of kelp.