Knock down to level up: Reframing RNAi for invertebrate ecophysiology.

Comparative ecophysiologists strive to understand physiological problems in non-model organisms, but molecular tools such as RNA interference (RNAi) are under-used in our field. Here, we provide a framework for invertebrate ecophysiologists to use RNAi to answer questions focused on physiological processes, rather than as a tool to investigate gene function. We specifically focus on non-model invertebrates, in which the use of other genetic tools (e.g., genetic knockout lines) is less likely. We argue that because RNAi elicits a temporary manipulation of gene expression, and resources to carry out RNAi are technically and financially accessible, it is an effective tool for invertebrate ecophysiologists. We cover the terminology and basic mechanisms of RNA interference as an accessible introduction for "non-molecular" physiologists, include a suggested workflow for identifying RNAi gene targets and validating biologically relevant gene knockdowns, and present a hypothesis-testing framework for using RNAi to answer common questions in the realm of invertebrate ecophysiology. This review encourages invertebrate ecophysiologists to use these tools and workflows to explore physiological processes and bridge genotypes to phenotypes in their animal(s) of interest.

A seasonal study on the microbiomes of Diploid vs. Triploid eastern oysters and their denitrification potential.

Oyster reefs are hotspots of denitrification mediated removal of dissolved nitrogen (N), however, information on their denitrifier microbiota is scarce. Furthermore, in oyster aquaculture, triploids are often preferred over diploids, yet again, microbiome differences between oyster ploidies are unknown. To address these knowledge gaps, farmed diploid and triploid oysters were collected over an annual growth cycle and analyzed using shotgun metagenomics and quantitative microbial elemental cycling (QMEC) techniques. Regardless of ploidy, Psychrobacter genus was abundant, with positive correlations found for genes of central metabolism, DNA metabolism, and carbohydrate metabolism. MAGs (metagenome-assembled genomes) yielded multiple Psychrobacter genomes harboring norB, narH, narI, and nirK denitrification genes, indicating their functional relevance within the eastern oysters. QMEC analysis indicated the predominance of carbon (C) and nitrogen (N) cycling genes, with no discernable patterns between ploidies. Among the N-cycling genes, the nosZII clade was overrepresented, suggesting its role in the eastern oyster's N removal processes.

Low-Level Dietary Clothianidin Exposure Preferentially Causes Prepupal Mortality of Monarch Butterflies (Danaus plexippus).

Data from prior research indicate the prepupal stage of the monarch butterfly life cycle is more sensitive to clothianidin exposure than the larval stage. A set of experiments was conducted to determine if the dietary clothianidin exposures that cause prepupal mortality are environmentally relevant. Monarch larvae were raised from egg to pupae on clothianidin-contaminated swamp milkweed plants (Asclepias incarnata). Larval growth as well as larval and prepupal survival were monitored throughout the experiments, in which the exposures ranged from 1.4 to 2793.1 ng/g leaf. Exposures of 5.4 to 46.9 ng/g leaf resulted primarily in prepupal mortality, whereas higher exposures of 1042.4 to 2793.1 ng/g leaf resulted exclusively in larval mortality, indicating the prepupal stage is more sensitive to clothianidin exposure than the larval stage. A median lethal concentration and a 10% lethal concentration of 37 and 6 ng/g leaf, respectively, were estimated for prepupal mortality. Both effect concentrations are within the range of clothianidin concentrations reported in leaves collected from wild milkweed plants, indicating prepupal mortality is an environmentally relevant effect. Environ Toxicol Chem 2024;00:1-6. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Novel ApeC-containing protein mediates the recognition and internalization of Vibrio splendidus in Apostichopus japonicus.

Pattern recognition receptors (PRRs) mediate the innate immune responses and play a crucial role in host defense against pathogen infections. Apextrin C-terminal (ApeC)-containing proteins (ACPs), a newly discovered class of PRRs specific to invertebrates, recognize pathogens through their ApeC domain as intracellular or extracellular effectors. However, the other immunological functions of ACPs remain unclear. In this study, a membrane-localized ACP receptor was identified in the sea cucumber Apostichopus japonicus (denoted as AjACP1). The ApeC domain of AjACP1, which was located outside of its cell membrane, exhibited the capability to recognize and aggregate Vibrio splendidus. AjACP1 was upregulated upon V. splendidus infection, internalizing into the cytoplasm of coelomocytes. AjACP1 overexpression enhanced the phagocytic activity of coelomocytes against V. splendidus, while knockdown of AjACP1 by RNA interfere inhibited coelomocyte endocytosis. Inhibitor experiments indicated that AjACP1 regulated coelomocyte phagocytosis through the actin-dependent endocytic signaling pathway. Further investigation revealed that AjACP1 interacted with the subunit of the actin-related protein 2/3 complex ARPC2, promoting F-actin polymerization and cytoskeletal rearrangement and thereby affecting the coelomocyte phagocytosis of V. splendidus via the actin-dependent endocytic signaling pathway. As a novel membrane PRR, AjACP1 mediates the recognition and phagocytic activity of coelomocytes against V. splendidus through the AjACP1-ARPC2-F-actin polymerization and cytoskeletal rearrangement pathway.

Different behavioral profiles between invasive and native nudibranchs: means for invasion success?

Behavior is predicted to be a primary determinant of the success of the invasion process during the early phases of colonization. Comparing invaders with sympatric native species may provide a good approach to unravel behavioral traits involved in an invasion process. In this study, we carried out an experimental simulation of the introduction and the acclimatization phase into a new environment and assessed the expression of activity, alertness, and habituation in an invasive Mediterranean population of the South African nudibranch Godiva quadricolor comparing its profiles with those of the sympatric Mediterranean native nudibranchs Cratena peregrina and Caloria quatrefagesi. Individuals of these 3 species were subjected to 3 behavioral tests: spontaneous activity, carried out in the introduction phase (immediately after sampling) and after a week of acclimatization; alert test, in which a potential threat was simulated by means of a tactile stimulus, and habituation test, in which the same alert test stimulus was repeated 5 times at 30-min intervals. The invasive G. quadricolor showed higher levels of exploration activity, thigmotaxis, alertness, and sensitization than the native species. These behavioral traits may represent pivotal drivers of the ongoing invasion process.

An ecohydrological approach to assess water provisioning and supporting ecosystem services in the Budhabalanga River Basin, India.

Rivers are vital and complex natural systems that provide a wide range of ecosystem services. This study presents a methodology for assessing the riverine provisioning and supporting ecosystem services, whose applicability has been demonstrated over the Budhabalanga River Basin of India. The Soil and Water Assessment Tool (SWAT) is used to generate streamflow time series at various ungauged sites, and then the streamflow is characterized for the evaluation of provisioning services. Further, the diversity and abundance of macroinvertebrates, along with the Lotic-invertebrate Index for Flow Evaluation (LIFE), is used to study the riverine supporting ecosystem services. The streams show intermittent behavior and strong seasonality for low flows, which limits the water availability, particularly during pre-monsoon season. The Baseflow Index (BFI) is greater than 0.6, indicating that groundwater contributes more than 60% of the total streamflow. Interestingly, despite the high BFI, the streams did not conform to the prevailing opinion that a greater baseflow contribution results in a later commencement of the low-flow period in the hydrological year. Furthermore, the study depicts significant variations in the diversity and abundance of the macroinvertebrates across the various sampling sites. However, the LIFE score across the sites remained consistent within a narrow range, i.e., 8 to 9, suggesting a steady supply of supporting ecosystem services. The results of the study can help the policymakers towards an informed decision making and the simplistic methodology proposed in this study can be replicated in other river basins for identifying vulnerable watersheds and prioritizing management actions.

Reproductive Capacity, but not Food Consumption, is Reduced by Continuous Exposure to Typical Genotoxic Stressor γ-Rays in the sentinel species Gammarus fossarum.

The long-term impacts of radiocontaminants (and the associated risks) for ecosystems are still subject to vast societal and scientific debate while wildlife is chronically exposed to various sources and levels of either environmental or anthropogenic ionizing radiation from the use of nuclear energy. The present study aimed to assess induced phenotypical responses in both male and female gammarids after short-term continuous γ-irradiation, acting as a typical well-characterized genotoxic stressor that can interact directly with living matter. In particular, we started characterizing the effects using standardized measurements for biological effects on few biological functions for this species, especially feeding inhibition tests, molting, and reproductive ability, which have already been proven for chemical substances and are likely to be disturbed by ionizing radiation. The results show no significant differences in terms of the survival of organisms (males and females), of their short-term food consumption which is linked to the general health status (males and females), and of the molting cycle (females). In contrast, exposure significantly affected fecundity (number of embryos produced) at the highest dose rates for irradiated females (51 mGy h-1) and males (5 and 51 mGy h-1). These results showed that, in gammarids, reproduction, which is a critical endpoint for population dynamics, is the most radiosensitive phenotypic endpoint, with significant effects recorded on male reproductive capacity, which is more sensitive than in females. Environ Toxicol Chem 2024;00:1-9. © 2024 SETAC.

Fire impacts on the biology of stream ecosystems: A synthesis of current knowledge to guide future research and integrated fire management.

Freshwater ecosystems host disproportionately high biodiversity and provide unique ecosystem services, yet they are being degraded at an alarming rate. Fires, which are becoming increasingly frequent and intense due to global change, can affect these ecosystems in many ways, but this relationship is not fully understood. We conducted a systematic review to characterize the literature on the effects of fires on stream ecosystems and found that (1) abiotic indicators were more commonly investigated than biotic ones, (2) most previous research was conducted in North America and in the temperate evergreen forest biome, (3) following a control-impact (CI) or before-after (BA) design, (4) predominantly assessing wildfires as opposed to prescribed fires, (5) in small headwater streams, and (6) with a focus on structural and not functional biological indicators. After quantitatively analyzing previous research, we detected great variability in responses, with increases, decreases, and no changes being reported for most indicators (e.g., macroinvertebrate richness, fish density, algal biomass, and leaf decomposition). We shed light on these seemingly contradicting results by showing that the presence of extreme hydrological post-fire events, the time lag between fire and sampling, and whether the riparian forest burned or not influenced the outcome of previous research. Results suggest that although wildfires and the following hydrological events can have dramatic impacts in the short term, most biological endpoints recover within 5-10 years, and that detrimental effects are minimal in the case of prescribed fires. We also detected that no effects were more often reported by BACI studies than by CI or BA studies, raising the question of whether this research field may be biased by the inherent limitations of CI and BA designs. Finally, we make recommendations to help advance this field of research and guide future integrated fire management that includes the protection of freshwater ecosystems.

Infrequent Long-Range Dispersal and Evolution of a Top Terrestrial Arthropod Predator in the Sub-Antarctic.

AbstractThe sub-Antarctic terrestrial ecosystems survive on isolated oceanic islands in the path of circumpolar currents and winds that have raged for more than 30 million years and are shaped by climatic cycles that surpass the tolerance limits of many species. Surprisingly little is known about how these ecosystems assembled their native terrestrial fauna and how such processes have changed over time. Here, we demonstrate the patterns and timing of colonization and speciation in the largest and dominant arthropod predators in the eastern sub-Antarctic: spiders of the genus Myro. Our results indicate that this lineage originated from Australia before the Plio-Pleistocenic glacial cycles and underwent an adaptive radiation on the Crozet archipelago, from where one native species colonized multiple remote archipelagos via the Antarctic circumpolar current across thousands of kilometers. The results indicate limited natural connectivity between terrestrial macroinvertebrate faunas in the eastern sub-Antarctic and partial survival of repeated glaciations in the Plio-Pleistocene. Furthermore, our findings highlight that by integrating arthropod taxa from multiple continents, the climatically more stable volcanic Crozet archipelago played a critical role in the evolution and distribution of arthropod life in the sub-Antarctic.

Establishing Silphids in the invertebrate DNA toolbox: a proof of concept.

Environmental DNA (eDNA) analyses are an increasingly popular tool for assessing biodiversity. eDNA sampling that uses invertebrates, or invertebrate DNA (iDNA), has become a more common method in mammal biodiversity studies where biodiversity is assessed via diet analysis of different coprophagous or hematophagous invertebrates. The carrion feeding family of beetles (Silphidae: Coleoptera, Latreille (1807)), have not yet been established as a viable iDNA source in primary scientific literature, yet could be useful indicators for tracking biodiversity in forested ecosystems. Silphids find carcasses of varying size for both food and reproduction, with some species having host preference for small mammals; therefore, iDNA Silphid studies could potentially target small mammal communities. To establish the first valid use of iDNA methods to detect Silphid diets, we conducted a study with the objective of testing the validity of iDNA methods applied to Silphids using both Sanger sequencing and high throughput Illumina sequencing. Beetles were collected using inexpensive pitfall traps in Alberta, Michigan in 2019 and 2022. We successfully sequenced diet DNA and environmental DNA from externally swabbed Silphid samples and diet DNA from gut dissections, confirming their potential as an iDNA tool in mammalian studies. Our results demonstrate the usefulness of Silphids for iDNA research where we detected species from the genera Anaxyrus, Blarina, Procyon, Condylura, Peromyscus, Canis, and Bos. Our results highlight the potential for Silphid iDNA to be used in future wildlife surveys.

Changes in the composition of invertebrate assemblages from wave-exposed intertidal mussel stands along the Nova Scotia coast, Canada.

Rocky intertidal habitats occur worldwide and are mainly characterized by primary space holders such as seaweeds and sessile invertebrates. Some of these organisms are foundation species, as they can form structurally complex stands that host many small invertebrates. The abundance of primary space holders is known to vary along coastlines driven directly or indirectly by environmental variation. However, it is less clear if the invertebrate assemblages associated to a foundation species may remain relatively unchanged along coastlines, as similar stands of a foundation species can generate similar microclimates. We examined this question using abundance data for invertebrate species found in mussel stands of a similar structure in wave-exposed rocky habitats at mid-intertidal elevations along the Atlantic coast of Nova Scotia (Canada). While the most abundant invertebrate species were found at three locations spanning 315 km of coastline, species composition (a combined measure of species identity and their relative abundance) differed significantly among the locations. One of the species explaining the highest amount of variation among locations (a barnacle) exhibited potential signs of bottom-up regulation involving pelagic food supply, suggesting benthic-pelagic coupling. The abundance of the species that explained the highest amount of variation (an oligochaete) was positively related to the abundance of their predators (mites), further suggesting bottom-up forcing in these communities. Overall, we conclude that species assemblages associated to structurally similar stands of a foundation species can show clear changes in species composition at a regional scale.

Ice in the intertidal: patterns and processes of freeze tolerance in intertidal invertebrates.

Many intertidal invertebrates are freeze tolerant, meaning that they can survive ice formation within their body cavity. Freeze tolerance is a fascinating trait, and understanding its mechanisms is important for predicting the survival of intertidal animals during extreme cold weather events. In this Review, we bring together current research on the ecology, biochemistry and physiology of this group of freeze-tolerant organisms. We first introduce the ecology of the intertidal zone, then highlight the strong geographic and taxonomic biases within the current body of literature on this topic. Next, we detail current knowledge on the mechanisms of freeze tolerance used by intertidal invertebrates. Although the mechanisms of freeze tolerance in terrestrial arthropods have been well-explored, marine invertebrate freeze tolerance is less well understood and does not appear to work similarly because of the osmotic differences that come with living in seawater. Freeze tolerance mechanisms thought to be utilized by intertidal invertebrates include: (1) low molecular weight cryoprotectants, such as compatible osmolytes and anaerobic by-products; (2) high molecular weight cryoprotectants, such as ice-binding proteins; as well as (3) other molecular mechanisms involving heat shock proteins and aquaporins. Lastly, we describe untested hypotheses, methods and approaches that researchers can use to fill current knowledge gaps. Understanding the mechanisms and consequences of freeze tolerance in the intertidal zone has many important ecological implications, but also provides an opportunity to broaden our understanding of the mechanisms of freeze tolerance more generally.

Corrigendum: Plastic biodegradation by in vitro environmental microorganisms and in vivo gut microorganisms of insects.

[This corrects the article DOI: 10.3389/fmicb.2022.1001750.].

Transgenesis enables mapping of segmental ganglia in the leech Helobdella austinensis.

The analysis of how neural circuits function in individuals and change during evolution is simplified by the existence of neurons identified as homologous within and across species. Invertebrates, including leeches, have been used for these purposes in part because their nervous systems comprise a high proportion of identified neurons, but technical limitations make it challenging to assess the full extent to which assumptions of stereotypy hold true. Here, we introduce Minos plasmid-mediated transgenesis as a tool for introducing transgenes into the embryos of the leech Helobdella austinensis (Spiralia; Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae). We identified an enhancer driving pan-neuronal expression of markers, including histone2B:mCherry, which allowed us to enumerate neurons in segmental ganglia. Unexpectedly, we found that the segmental ganglia of adult transgenic H. austinensis contain fewer and more variable numbers of neurons than in previously examined leech species.

Characterization of Springtail (Arrhopalites caecus) for Use in Soil Ecotoxicity Testing.

Springtails (subclass: Collembola) represent one of the most extensively studied invertebrate groups in soil ecotoxicology. This is because of their ease of laboratory culture, significant ecological role, and sensitivity to environmental contaminants. Folsomia candida (family: Isotomidae) is a globally widespread parthenogenetic species that is prevalent in laboratory toxicity testing with springtails. Conversely, Arrhopalites caecus (family: Arrhopalitidae), a parthenogenic globular springtail species, remains untested in soil ecotoxicology. This species is found in diverse habitats, including cave systems and forest leaf litter, and has a global distribution. The sensitivity of A. caecus to environmental contaminants, such as neonicotinoid insecticides, as well as its life history and optimal culturing conditions, are largely unknown. The present study describes the establishment of a pure A. caecus laboratory culture and characterization of its life cycle and culturing conditions. We assessed the sensitivity of A. caecus to various insecticides, including exposures to the neonicotinoid thiamethoxam in soil and through a novel feeding assay as well as to clothianidin and cyantraniliprole in spiked soil exposures. In 7- and 14-day exposures to thiamethoxam in agricultural soil, the 50% lethal concentration (LC50) values were determined to be 0.129 mg/kg dry weight and 0.010 mg/kg dry weight, respectively. The 14-day LC50 for exposure to thiamethoxam via spiked food was determined to be 0.307 mg/kg dry weight. In addition, the 28-day 50% effect concentration for inhibition of juvenile production from cyantraniliprole exposure in the same soil type was 0.055 mg/kg dry weight. Challenges encountered in using this species included susceptibility to mite infestation and low adult survival rates in the 28-day cyantraniliprole test. Environ Toxicol Chem 2024;43:1820-1835. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

A new spider species of Belisana Thorell, 1898 (Araneae, Pholcidae) from Guizhou Province, south-western China.

Background: China exhibits remarkable diversity of the spider genus Belisana Thorell, 1898, with 62 species recorded to date. However, the largest number of Belisana species was found in Yunnan Province (23 ssp.), while only seven species were found in Guizhou Province. New information: In this paper, Belisanawangchengi sp. nov. as a new species is described from Guizhou Province, China.

Ribbon worms (phylum Nemertea) from Bodega Bay, California: A largely undescribed diversity.

The diversity of nemerteans along the Pacific coast of the United States is regarded as well characterized, but there remain many cryptic, undescribed, and "orphan" species (those known only in their larval form). Recent sampling of nemerteans in Oregon and Washington has begun to fill in these taxonomic gaps, but nemertean diversity in California has received relatively little attention over the past 60 years. During the summers of 2019 and 2020, nemertean specimens were collected from 20 locations in the Bodega Bay region of northern California, USA, including rocky intertidal shores, sandy beaches, mudflats, and other habitats. Based on morphological assessment and DNA sequence analysis (partial Cytochrome Oxidase I and 16S rRNA genes), our surveys identified 34 nemertean species. Only 13 of these (38%) can be confidently assigned to described species. Another 11 represent species that are new to science, including members of the genera Riserius, Nipponnemertes, Poseidonemertes, Zygonemertes, Nemertellina, Oerstedia, and three species of uncertain affiliation. The remaining ten species include undescribed or cryptic species of uncertain status that have been found previously along the Pacific Coast of North America. Our surveys also document extensions of known geographic ranges for multiple species, including the first records in California of Antarctonemertesphyllospadicola, Cephalothrixhermaphroditica, and Maculauraoregonensis. This is the first report of the genus Nemertellina in the northeast Pacific and Riserius in California. Overall, our findings highlight how much remains to be learned about the diversity and distribution of nemerteans in the northeast Pacific.

Eight new spider species of Belisana Thorell, 1898 (Araneae, Pholcidae), with an updated overview of Belisana species from Yunnan, China.

In this study, eight new species are described from the subtropical parts of Yunnan Province in southwestern China: Belisanahonghe Zhang, Li & Yao, sp. nov. (♂♀), B.jiuxiang Zhang, Li & Yao, sp. nov. (♂♀), B.lincang Zhang, Li & Yao, sp. nov. (♂♀), B.luxi Zhang, Li & Yao, sp. nov. (♂♀), B.tengchong Zhang, Li & Yao, sp. nov. (♂♀), B.tongi Zhang, Li & Yao, sp. nov. (♂♀), B.yongsheng Zhang, Li & Yao, sp. nov. (♂), and B.yunnan Zhang, Li & Yao, sp. nov. (♂♀). They add up to a total of 31 Belisana species from Yunnan in an updated list provided in this paper.

Amphioxus (Branchiostoma lanceolatum) in the North Adriatic Sea: ecological observations and spawning behavior.

The European amphioxus (Branchiostoma lanceolatum) is a member of the chordate subphylum Cephalochordata, and, as such, a key model organism for providing insights into the origin and evolution of vertebrates. Despite its significance and global distribution, detailed characterizations of natural populations of cephalochordates are still very limited. This study investigates the abundance, habitat, and spawning behavior of amphioxus in the North Adriatic Sea. Across 32 sampled sites, adult amphioxus were consistently present, reaching densities exceeding 300 individuals m- 2. DNA barcoding confirmed the species as B. lanceolatum, and environmental analyses revealed an amphioxus preference for slightly gravelly sand with low silt content and a correlation between amphioxus density and the presence of specific macroinvertebrate taxa. Remarkably, the amphioxus population was breeding in early spring and possibly late fall, in contrast to the typical late spring/early summer spawning season described for other populations of European amphioxus. Amphioxus adults kept in captivity maintained the spawning seasonality of their place of origin, suggesting the possibility of extending the overall spawning season of European amphioxus in laboratory settings by exploiting populations from diverse geographic origins. This study thus expands our understanding of B. lanceolatum ecology and reproduction in the Mediterranean Sea, emphasizing the role of the North Adriatic Sea as a substantial reservoir.

Artificial light and cloud cover interact to disrupt celestial migrations at night.

The growth of human activity and infrastructure has led to an unprecedented rise in the use of Artificial Light at Night (ALAN) with demonstrable impacts on ecological communities and ecosystem services. However, there remains very little information on how ALAN interacts with or obscures light from celestial bodies, which provide vital orientating cues in a number of species. Furthermore, no studies to date have examined how climatic conditions such as cloud cover, known to influence the intensity of skyglow, interact with lunar irradiance and ALAN over the course of a lunar cycle to alter migratory abilities of species. Our night-time field study aimed to establish how lunar phase and climatic conditions (cloud cover) modulate the impact of ALAN on the abundance and migratory behaviour of Talitrus saltator, a key sandy beach detritivore which uses multiple light associated cues during nightly migrations. Our results showed that the number and size of individuals caught decreased significantly as ALAN intensity increased. Additionally, when exposed to ALAN more T. saltator were caught travelling parallel to the shoreline, indicating that the presence of ALAN is inhibiting their ability to navigate along their natural migration route, potentially impacting the distribution of the population. We found that lunar phase and cloud cover play a significant role in modifying the impact of ALAN, highlighting the importance of incorporating natural light cycles and climatic conditions when investigating ALAN impacts. Critically we demonstrate that light levels as low as 3 lx can have substantial effects on coastal invertebrate distributions. Our results provide the first evidence that ALAN impacted celestial migration can lead to changes to the distribution of a species.