Successful Metamorphosis Research Articles

Microfluidics for hydrodynamic investigations of sand dollar larvae

The life cycle of most marine invertebrates includes a planktonic larval stage before metamorphosis to bottom-dwelling adulthood. During the larval stage, ciliary-mediated activity enables feeding (capturing unicellular algae) and transporting materials (e.g., oxygen) required for the larva’s growth, development, and successful metamorphosis. Investigating the underlying hydrodynamics of the ciliary activities is valuable for addressing fundamental biological questions (e.g., phenotypic plasticity) and advancing engineering applications (e.g., biomimetic design). We combined microfluidics and fluorescence microscopy as a miniaturized particle image velocimetry approach to study ciliary-mediated hydrodynamics during suspension feeding in sand dollar larvae (Dendraster excentricus). First, feasibility was confirmed by examining the underlying hydrodynamics (ciliary-mediated vortex patterns) for low- and high-fed larvae. Next, ciliary hydrodynamics were tracked from 11 days post-fertilization (DPF) to 20 DPF for 21 low-fed larvae. Microfluidics enabled the examination of baseline activities (without external flow) and behaviors in the presence of environmental cues (external flow). A library of qualitative vortex patterns and quantitative hydrodynamics (velocity and vorticity profiles) was generated. Velocities were used to examine the role of ciliary activity in transporting materials. Given the laminar flow and the viscosity-dominated environments surrounding the larvae, overcoming the diffusive boundary layer is critical. Péclet number analysis for oxygen transport suggested that ciliary velocities help overcome the diffusion-dominated transport. The approach was used to examine the hydrodynamics of two additional marine larvae (P. helianthoides and S. purpuratus). Microfluidics provided a scalable and accessible approach for investigating the ciliary hydrodynamics of marine organisms.

Dead ringer acts as a major regulator of juvenile hormone biosynthesis in insects.

In holometabolous insects, proper control of the production of juvenile hormone (JH), which maintains larval traits, is crucial for successful metamorphosis. JH is produced specifically in the corpora allata (CA) via the functioning of a set of JH biosynthetic enzymes (JHBEs). Expression of JHBE genes in the CA is coordinated except for JH acid methyltransferase (JHAMT), which functions in the last step of JH biosynthesis. Here, we sought to determine the mechanism that enables this coordinated expression, assuming the presence of a central regulator of JHBE genes. Comparison of transcriptomes in the CA during active and inactive stages revealed the presence of 3 transcription factors, whose expression patterns matched those of JHBE genes. We propose that one of these, Dead ringer (Dri), is the central up-regulator of CA-specific JHBE genes including JHAMT, based on the following findings: (ⅰ) Knockdown of Dri in the larvae caused precocious metamorphosis, which was rescued by the exogenous application of JH analog, and (ⅱ) knockdown of Dri decreased the expression of most CA-specific JHBE genes examined. Furthermore, RNAi-based reverse genetics indicated that Dri works most upstream in the control of CA-specific JHBE genes, and that shutdown of JHAMT, which occurs independent of other JHBE genes prior to the onset of metamorphosis, can be hypothetically explained by the presence of an unidentified repressor. Our study suggests that Dri, which has been known to regulate embryonic development in a wide range of animals, is conferred a new role in holometabolous insects, i.e. central regulation of CA-specific JHBE genes.

Model estimation of eastern oyster larval performance from food quantity and quality measures in western Mississippi Sound

Oyster Crassostrea virginica population recovery is critical in degraded estuarine systems, such as Mississippi Sound, USA, where repeated mass mortality events have depleted local oyster stocks. Owing to multiple recent die-offs, the western Mississippi Sound oyster population is recruitment-limited; population growth is constrained by the entry of new individuals into the extant population. Therefore, oyster recovery requires an adequate supply of larvae capable of timely development, growth, and successful metamorphosis. Larval performance and settlement potential are influenced by ambient temperature, salinity, and food supply. Food quantity is important to larvae, but so is food quality, as larvae require a balanced diet of lipids, proteins, and carbohydrates to develop and survive through metamorphosis. In this study, in situ environmental and food conditions during the 2021 and 2022 spawning seasons from 7 oyster reefs in western Mississippi Sound were integrated into an established biochemically based larval performance model to estimate periods facilitative of successful metamorphosis. In 2021, model-estimated larval survivorship was suppressed through much of the spawning season by prolonged, extremely low salinity (<5 ppt) and inadequately balanced food supply. Higher seasonal salinity and more balanced food composition increased model-estimated larval survivorship in 2022, despite lower total food content, suggesting larval performance was primarily governed by the quality of available food. Model-estimated settlement windows were compared to settlement windows derived from concomitant field observations of recruitment. Strong agreement between model-estimated and observed settlement windows validates the effectiveness of the model and informs on the underlying causes of recruitment limitation in western Mississippi Sound.

Integrative proteome and metabolome analyses reveal molecular basis of the tail resorption during the metamorphic climax of Nanorana pleskei.

During the metamorphosis of anuran amphibians, the tail resorption process is a necessary and crucial change. One subject that has received relatively little or no attention is the expression patterns of proteins and metabolites in the different tail portions during metamorphosis, especially in highland amphibians. The mechanisms of tail resorption in three portions (the tip, middle and root) of the tail were investigated in N. pleskei G43 tadpole based on two omics (proteomic and metabolomic). Integrin αVβ3 was found to be high expressed in the distal portion of the tail, which could improve the sensitiveness to thyroid hormones in the distal portion of the tail. Muscle regression displayed a spatial pattern with stronger regression in distal and weaker one in proximal portion. Probably, this stronger regression was mainly performed by the proteases of proteasome from the active translation by ribosomes. The suicide model and murder model coexisted in the tail resorption. Meanwhile, fatty acids, amino acids, pyrimidine, and purine which derived from the breakdown of tissues can be used as building blocks or energy source for successful metamorphosis. Our data improved a better comprehension of the tail resorption mechanisms underlying the metamorphism of N. pleskei tadpole through identifying important participating proteins and metabolites.

Gross morphology and adhesion-associated physical properties of Drosophila larval salivary gland glue secretion

One of the major functions of the larval salivary glands (SGs) of many Drosophila species is to produce a massive secretion during puparium formation. This so-called proteinaceous glue is exocytosed into the centrally located lumen, and subsequently expectorated, serving as an adhesive to attach the puparial case to a solid substrate during metamorphosis. Although this was first described almost 70 years ago, a detailed description of the morphology and mechanical properties of the glue is largely missing. Its main known physical property is that it is released as a watery liquid that quickly hardens into a solid cement. Here, we provide a detailed morphological and topological analysis of the solidified glue. We demonstrated that it forms a distinctive enamel-like plaque that is composed of a central fingerprint surrounded by a cascade of laterally layered terraces. The solidifying glue rapidly produces crystals of KCl on these alluvial-like terraces. Since the properties of the glue affect the adhesion of the puparium to its substrate, and so can influence the success of metamorphosis, we evaluated over 80 different materials for their ability to adhere to the glue to determine which properties favor strong adhesion. We found that the alkaline Sgs-glue adheres strongly to wettable and positively charged surfaces but not to neutral or negatively charged and hydrophobic surfaces. Puparia formed on unfavored materials can be removed easily without leaving fingerprints or cascading terraces. For successful adhesion of the Sgs-glue, the material surface must display a specific type of triboelectric charge. Interestingly, the expectorated glue can move upwards against gravity on the surface of freshly formed puparia via specific, unique and novel anatomical structures present in the puparial’s lateral abdominal segments that we have named bidentia.

The role of adaptive resistance in a widespread freshwater mussel species

Abstract Organism features, such as size, weight, sex and age, among others, can influence the success of parasitism. For species that depend on a host to complete their life cycle, such as freshwater mussels (Bivalvia, Unionida), understanding how these traits influence parasitism can help guide management actions aimed at improving their reproductive success, ultimately benefitting their conservation. In this study, the physiological compatibility between the duck mussel Anodonta anatina and its host fish was investigated in a simultaneous infestation experiment on four (three native and one non‐native) fish species using sympatric and allopatric host strains. The success of glochidia metamorphosis in juvenile mussels was evaluated as a function of host species, strain and fish length. Successful metamorphosis was achieved in almost all fish species tested and differences were detected between allopatric and sympatric strains, with higher values found in allopatric hosts. Allopatric strains of primary hosts produce more juveniles than marginal hosts and non‐native species. In addition, larger fish have lower rates of metamorphosis. The results confirm recent findings showing high variation in the metamorphosis success of A. anatina glochidia among different native and non‐native hosts, both in the natural environment and in artificial laboratory infestations. Allopatric hosts were more suitable for encystment, demonstrating potential adaptive immunity, not yet observed in A. anatina. Nevertheless, A. anatina may temporarily exploit the naivety of smaller fish regardless of strain. Overall, the results suggest that mussel propagation, reproduction and reintroduction efforts should consider the mechanisms that influence adaptive immunity in fish, including differences in compatibility within and between populations of sympatric and allopatric host strains. This situation should be taken into account, as successful metamorphosis is an important factor in the selection of hosts for juvenile development, especially in efforts to propagate endangered mussel species.

Metabolic and Developmental Changes in Insects as Stress-Related Response to Electromagnetic Field Exposure

(1) Background: The growing ubiquity of electromagnetic fields (EMF) due to rapid technological progress raises concerns about potential health implications. While laboratory experiments have generated inconclusive findings about adverse effects, EMFs have demonstrated efficacy in magnetotherapy. Earlier studies indicate that an EMF can trigger stress responses in organisms, the outcomes of which are dependent on the intensity of the EMF. (2) Methods: This study aims to explore the effects of extremely low-frequency EMF (50 Hz, 1 mT, or 7 mT) on metamorphosis and metabolism rates, which are indicators of stress, across different developmental stages of Tenebrio molitor, including adults, pupae, and larvae. (3) Results: Our findings reveal that exposure to EMF leads to accelerated weight loss, increased adult metabolism, and higher mortality; however, EMF exposure appears to have no impact on sugar levels or the rate and success of metamorphosis. Notably, significant changes were only observed under the influence of a strong EMF (7 mT), while the weaker EMF (1 mT) did not yield statistically significant outcomes. (4) Conclusion: The obtained results suggest that an extremely low-frequency EMF can be considered a stressor, with its effects contingent upon the specific parameters of exposure and the developmental stage of the experimental model.

Steroid hormone-dependent changes in trehalose physiology in the silkworm, Bombyx mori.

Holometabolous insects undergo metamorphosis to reconstruct their body to the adult form during pupal period. Since pupae cannot take any diets from the outside because of a hard pupal cuticle, those insects stock up on nutrients sufficient for successful metamorphosis during larval feeding period. Among those nutrients, carbohydrates are stored as glycogen or trehalose, which is the major blood sugar in insects. The hemolymph trehalose is constantly high during the feeding period but suddenly decreases at the beginning of the prepupal period. It is believed that trehalase, which is a trehalose-hydrolyzing enzyme, becomes highly active to reduce hemolymph trehalose level during prepupal period. This change in the hemolymph trehalose level has been interpreted as the physiological shift from storage to utilization of trehalose at that stage. Although this shift in trehalose physiology is indispensable for energy production required for successful metamorphosis, little is known on the regulatory mechanisms of trehalose metabolism in accordance with developmental progress. Here, we show that ecdysone, an insect steroid hormone, plays essential roles in the regulation of soluble trehalase activity and its distribution in the midgut of silkworm, Bombyx mori. In the end of larval period, soluble trehalase was highly activated in the midgut lumen. This activation was disappeared in the absence of ecdysone and also restored by ecdysone administration. Our present results suggest that ecdysone is essentially required for the changes in the function of the midgut on trehalose physiology as development progresses.

Effects of the neonicotinoid insecticides thiamethoxam and imidacloprid on metamorphosis of the toad Rhinella arenarum at environmentally-relevant concentrations

ABSTRACT The goal of the present study was to examine the effects of environmentally relevant concentrations of the neonicotinoid insecticides thiamethoxam and imidacloprid on the metamorphosis of the toad Rhinella arenarum. Tadpoles were exposed from stage 27 until completion of metamorphosis to concentrations of thiamethoxam ranging between 1.05 and 1050 µg/L and concentrations of imidacloprid varying between 3.4 and 3400 µg/L. The two neonicotinoids were found to act differently at the range of concentrations tested. Thiamethoxam did not markedly alter the final % tadpoles completing metamorphosis but extended by 6–20 days the time needed for tadpoles to complete metamorphosis. The extra number of days required to reach metamorphosis was concentration-dependent between 1.05 and 100.5 µg/L, and then stable at 20 days between 100.5 and 1005 µg/L. In contrast, imidacloprid did not significantly interfere with the overall time needed to complete metamorphosis but decreased success of metamorphosis at 3400 µg/L, the highest concentration tested. Both neonicotinoid concentrations did not markedly alter body size and weight of the newly metamorphosed toads. With a lowest observed effect concentration (LOEC) of 1.05 µg/L, thiamethoxam may be more likely to impact tadpole development in the wild compared to imidacloprid, which was without any apparent effect at concentrations up to 340 µg/L (no-observed effect concentration or NOEC). As the influence of thiamethoxam was triggered after tadpoles had reached Stage 39, when metamorphosis is strictly dependent upon thyroid hormones, this observed effect is attributed to result from actions of this neonicotinoid insecticide on the hypothalamic-pituitary-thyroid axis.

Alterations to unionid transformation during agricultural and urban contaminants of emerging concern exposures

Highly imperiled unionids have a complex life cycle including the metamorphosis of an obligate parasite life stage, larval glochidia, to the juvenile stage. Despite the known vulnerabilities of both glochidia and juveniles to pollutants, little is known on how metamorphosis success may be affected by chemical stress. Disruption of the transformation process in which glochidia encyst on the gills of a host fish, could lead to lowered recruitment and population declines. Transformation rates of Lampsilis cardium on host fish Micropterus salmoides were empirically derived from experimental exposures to low, medium, or high concentrations of an agricultural or urban mixture of contaminants of emerging concern (CECs) over two exposure durations. Transformation was characterized by: (1) a zero-inflated Poisson general linear mixed effects model to compare difference in transformation between exposure durations and (2) time response curves to describe the transformation curve using long-term exposure data. Lampsilis cardium transformation was similar between exposure durations. When compared to controls, CEC stress significantly reduced juvenile production (p « 0.05) except for the agricultural medium treatment and tended to increased encapsulation duration which while statistically insignificant (p = 0.16) may have ecological relevancy. Combining the empirically derived reduction of transformation rates with parameters values from the literature, a Lefkovich stage-based population model predicted strong declines in population size of L. cardium for all treatments if these results hold in nature. Management focus on urban CECs may lead to best conservation efforts though agricultural CECs may also have a concentration dependent impact on transformation and therefore overall recruitment and conservation success.

Genome-wide chromatin accessibility and gene expression profiling during flatfish metamorphosis

Metamorphosis is a widely studied post-embryonic process in which many tissues undergo dramatic modifications to adapt to the new adult lifestyle. Flatfishes represent a good example of metamorphosis in teleost fishes. During metamorphosis of flatfish, organ regression and neoformation occur, with one of the most notable changes being the migration of one of the eyes to the other side of the body. In order to create a useful and reliable tool to advance the molecular study of metamorphosis in flatfish, we generated a chromatin accessible atlas as well as gene expression profile during four developmental stages ranging from a phylotypic to a post-metamorphic stage. We identified 29,019 differentially accessible chromatin regions and 3,253 differentially expressed genes. We found stage-specific regulatory regions and gene expression profiles, supporting the quality of the results. Our work provides strongly reproducible data for further studies to elucidate the regulatory elements that ensure successful metamorphosis in flatfish species.

Spawning and larval development of two tropical cowries (Gastropoda: Cypraeidae), Cypraea tigris and Mauritia arabica under laboratory conditions.

The spawning and larval culture of cowrie (family Cypraeidae) are both difficult and little known, in part due to the long planktonic period of most species. In this study, we describe the captive spawning behaviour and larval development of two tropical cowrie species, Cypraea tigris and Mauritia arabica. Both species brooded over their egg masses before hatching occurred and larvae were collected for culture under laboratory conditions. The brooding period for C. tigris was between 7 and 17 days, and freshly hatched veligers were approximately 200-240 µm in size. Cypraea tigris larvae were reared for up to 37 days in culture but did not achieve successful settlement. The brooding period for M. arabica was between 7 and 10 days, and hatched veligers were approximately 160-205 µm in size. The first settled juvenile M. arabica was observed at 70 days post-hatch. Our findings from this study represent the first comprehensive documentation of successful metamorphosis of Cypraeidae larvae, particularly M. arabica, into early-stage juvenile.

Temperature and feeding frequency impact the survival, growth, and metamorphosis success of Solea solea larvae.

Human-induced climate change impacts the oceans, increasing their temperature, changing their circulation and chemical properties, and affecting marine ecosystems. Like most marine species, sole has a biphasic life cycle, where one planktonic larval stage and juvenile/adult stages occur in a different ecological niche. The year-class strength, usually quantified by the end of the larvae stage, is crucial for explaining the species' recruitment. We implemented an experimental system for rearing larvae under laboratory conditions and experimentally investigated the effects of temperature and feeding frequencies on survival, development (growth), and metamorphosis success of S. solea larvae. Specific questions addressed in this work include: what are the effects of feeding regimes on larvae development? How does temperature impact larvae development? Our results highlight that survival depends on the first feeding, that the onset of metamorphosis varies according to rearing temperature and that poorly fed larvae take significantly longer to start (if they do) metamorphosing. Moreover, larvae reared at the higher temperature (a +4°C scenario) showed a higher incidence in metamorphosis defects. We discuss the implications of our results in an ecological context, notably in terms of recruitment and settlement. Understanding the processes that regulate the abundance of wild populations is of primary importance, especially if these populations are living resources exploited by humans.

Synergistic survival-related effects of larval exposure to an aquatic pollutant and food stress get stronger during and especially after metamorphosis and shape fitness of terrestrial adults

To improve the ecological risk assessment of aquatic pollutants it is needed to study their effects not only in the aquatic larval stage, but also in the terrestrial adult stage of the many animals with a complex life cycle. This remains understudied, especially with regard to interactive effects between aquatic pollutants and natural abiotic stressors. We studied effects of exposure to the pesticide DNP (2,4-Dinitrophenol) and how these were modulated by limited food availability in the aquatic larvae, and the possible delayed effects in the terrestrial adults of the damselfly Lestes viridis. Our results revealed that DNP and low food each had large negative effects on the life history, behaviour and to a lesser extent on the physiology of not only the larvae, but also the adults. Food limitation magnified the negative effects of DNP as seen by a strong decline in larval survival, metamorphosis success and adult lifespan. Notably, the synergism between the aquatic pollutant and food limitation for survival-related traits was stronger in the non-exposed adults than in the exposed larvae, likely because metamorphosis is stressful itself. Our results highlight that identifying effects of aquatic pollutants and synergisms with natural abiotic stressors, not only in the aquatic larval but also in the terrestrial adult stage, is crucial to fully assess the ecological impact of aquatic pollutants and to reveal the impact on the receiving terrestrial ecosystem through a changed aquatic-terrestrial subsidy.

August Meditations

translated by Mina Karavanta, Prof of English Literature at Athens University 1. If a man in his forties is still drawing seas and dovecotes, if in his thought is reflected a sun more transparent, more lucid than the sun of reality, if the word “Amorgos” is not just the mask of a fleeting, adolescent memory, then between the poem of desire and the poem of necessity real loss is throbbing. 2. Prologues have been consumed. They cannot always substitute the topic. He must decide whether he can hold on to this absolute idea even if he has ceased to believe in its power. 3. Successive metamorphoses of paradise. The eye tries to interpret the enigma of beauty while Delos is slowly emerging in the horizon. Summer feels like an eternity. The poem begins to invent itself the moment he turns his face to the light. (The moment the imagination, freed from the sensation of the blazing white,

Sensitivity of Anguilliformes leptocephali to metamorphosis stimulated by thyroid hormone depends on larval size and metamorphic stage

Metamorphosis of teleosts including Anguilliformes is well known to be induced by thyroid hormone (TH), although the underlying mechanism is not fully understood. In this study, we investigated the experimental conditions needed to induce normal metamorphosis in artificially spawned Japanese eel (Anguilla japonica), including initial larval size, TH concentration, and timing of TH immersion. Around 37 mm TL was found to be the minimum size of larvae that underwent successful metamorphosis induced by l-thyroxine (T4); notably, smaller larvae did not show increased expression of TH receptors in response to T4, suggesting that small leptocephali are not sufficiently responsive to TH. Furthermore, successful completion of metamorphosis depended on sensitivity to TH, which changed with metamorphic stage; for example, prolonged exposure to higher TH concentrations led to morphological defects. Collectively, these results reveal that the induction of metamorphosis by TH is dependent on larval size, and that the concentration of TH must be adjusted in line with metamorphic stage to achieve successful progression of metamorphosis. Our findings will contribute to improving production technology in the aquaculture of Japanese eels by facilitating the earlier induction of metamorphosis in artificial leptocephali.

The parasitic travel of Margaritifera margaritifera in Atlantic salmon gills: from glochidium to post-larva

The larval development of the endangered freshwater mussel Margaritifera margaritifera (L.) represents one of the most unique parasitism among naiads, in which larva parasite the fish gills for several months. Despite the importance of this parasitic phase to successfully culture the freshwater mussel, the larval morphogenesis remains understudied. To describe the parasitic larval development and metamorphosis, Atlantic salmon (Salmo salar L.) were exposed to glochidia, sampled periodically to visualize the gills by stereomicroscopy and light microscopy and results were summarized throughout three developmental stages. Once attached to the fish gills, glochidia changed their morphology within the first days and acquired an intermediate stage termed mushroom larva due to the presence of the mushroom body and the zip membrane, both structures are transitory and distinctive of this long-lasting parasitism. The zip membrane, located at the valve cleft, may play a unique role in the isolation and acquisition of non-particulate nutrients from the fish, while the mushroom body of the mantle accumulates abundant intracytoplasmic lipid droplets. After 200 days, a successful metamorphosis was evidenced by the formation of a complete set of post-larval organs, pointing to the acquisition of different functionality, which will be essential for the settlement and deposit-feeding into the riverbed. Among the post-larval organs, the byssal complex of the post-larval foot was described for the first time at the end of the parasitic stage of naiads. In conclusion, this study provides an overview of the larval morphogenesis of M. margaritifera, from glochidium to post-larva, essential for understanding the parasitic interaction between the freshwater mussel larva and the fish host. Moreover, the morphological techniques and the hallmarks described might be applicable to optimize and monitor the larval developmental status during one of the most critical stages of the captive breeding programmes of endangered freshwater mussels.

A marine probiotic treatment against the bacterial pathogen Vibrio coralliilyticus to improve the performance of Pacific (Crassostrea gigas) and Kumamoto (C. sikamea) oyster larvae

Oyster larvae reared in hatcheries on the U.S. West coast often experience severe Vibrio coralliilyticus-related mortalities early in their development. Current treatment options for these molluscs are either not available or feasible; however, for decades, probiotics have been successfully used in finfish and crustacean shellfish culture. Consequently, the objectives of this work were to 1) isolate marine bacteria from oysters and evaluate their protective activity against Vibrio coralliilyticus infection of Pacific oyster (Crassostrea gigas) larvae, and 2) to determine the long-term effects of probiotic additions on growth and metamorphosis of larval Pacific and Kumamoto oysters (C. sikamea). A combination of three probiotic strains applied once 24 h post-fertilization was more effective in improving survival of larval C. gigas exposed to lethal concentrations of V. coralliilyticus strain RE22, compared with separate additions of individual probiotics. In addition, a single application of the probiotic combination to one-day-old larvae increased the larval metamorphosis success of C. sikamea and both the Midori and Myiagi stocks of C. gigas. These results suggest that probiotics are effective at preventing bacterial infections and can significantly improve performance of oyster larvae, using a single application early in their development.

First Evidence of Feeding-Induced RNAi in Banana Weevil via Exogenous Application of dsRNA.

Simple SummaryRNA interference (RNAi), a conserved mechanism in eukaryotic organisms, is initiated by the presence of double-stranded RNA (dsRNA) in the cells, regulating the expression of specific genes by degradation of their mRNA, and it is known as post-transcriptional gene-silencing (PTGS). RNAi can be employed to develop species-specific biopesticides where dsRNA is delivered to a pest insect via the oral route. Banana weevil (Cosmopolites sordidus) is the most devastating pest of banana and plantain worldwide, yet current control measures are neither effective, sustainable, nor environmentally sound. This study reports an artificial diet-based multi-well plate method and the first findings on the potential application of biotechnology in the control of the banana weevil using dietary RNAi, which would arguably provide the most sustainable and practical method for dsRNA delivery. A selection of target genes with effective RNAi leading to high and rapid mortality is presented. These results indicate the potential of RNAi-mediated management to suppress C. sordidus efficiently in the banana crop.Banana weevil (Cosmopolites sordidus) is the most devastating pest of banana and plantain worldwide, yet current control measures are neither effective, sustainable, nor environmentally sound, and no resistant farmer-preferred cultivars are known to date. In this paper, we examined the ability to induce RNA interference (RNAi) in the banana weevil via feeding. We first developed an agar- and banana corm (rhizome) flour-based artificial diet in a multi-well plate setup that allowed the banana weevils to complete their life cycle from egg through the larval instars to the pupal stage in an average period of 53 days. Adults emerged about 20 days later. The artificial diet allowed the tunneling and burrowing habits of the larvae and successful metamorphosis up to adult eclosion. Adding dsRNA for laccase2 to the artificial diet resulted in albino phenotypes, confirming gene-silencing. Finally, C. sordidus was fed with dsRNA against a selection of essential target genes: snf7, rps13, mad1, vha-a, vha-d, and lgl for a period of 45 days. 100% mortality within 9–16 days was realized with dssnf7, dsrps13, and dsmad1 at 200 ng/mL artificial diet, and this corresponded to a strong reduction in gene expression. Feeding the dsRNA targeting the two vha genes resulted in 100% mortality after about 3–4 weeks, while treatment with dslgl resulted in no mortality above the dsgfp-control and the water-control. Our results have implications for the development of RNAi approaches for managing important crop pests, in that banana weevils can be controlled based on the silencing of essential target genes as snf7, rps13, and mad1. They also highlight the need for research into the development of RNAi for banana protection, eventually the engineering of host-induced gene-silencing (HIGS) cultivars, given the high RNAi efficacy and its species-specific mode of action, adding the RNAi approach to the armory of integrated pest management (IPM).

Single and combined effects of microplastics, pyrethroid and food resources on the life-history traits and microbiome of Chironomus riparius

There is growing evidence of widespread contamination of freshwater ecosystems with microplastics. However, the effects of chronic microplastic ingestion and its interaction with other pollutants and stress factors on the life-history traits and the host-microbiome of aquatic invertebrates are not well understood. This study investigates the effects of exposure to sediment spiked with 1 μm polystyrene-based latex microplastic spheres, an environmentally realistic concentration of a pyrethroid pesticide (esfenvalerate), and a combination of both treatments on the life-history traits of the benthic-dwelling invertebrate, Chironomus riparius and its microbial community. The chironomid larvae were also exposed to two food conditions: abundant or limited food in the sediment, monitored for 28 and 34 days respectively. The microplastics and esfenvalerate had negative effects on adult emergence and survival, and these effects differed between the food level treatments. The microbiome diversity was negatively affected by the exposure to microplastics, while the relative abundances of the four top phyla were significantly affected only in the high food level treatment. Although the combined exposure to microplastics and esfenvalerate showed some negative effects on survival and emergence, there was little evidence for synergistic effects when compared to the single exposure. The food level affected all life-history traits and the microbiota, and lower food levels intensified the negative effects of the exposure to microplastics, esfenvalerate and their combination. We argue that these pollutants can affect crucial life-history traits such as successful metamorphosis and the host-microbiome. Therefore, it should be taken into consideration for toxicological assessment of pollutant acceptability. Our study highlights the importance of investigating possible additive and synergic activities between stressors to understand the effects of pollutants in the life story traits and host-microbiome.