Haemolymph transfusions transfer heritable learned novel odour preferences to naive larvae of Bicyclus anynana butterflies.

The mechanisms whereby environmental experiences of parents are transmitted to their offspring to impact their behaviour and fitness are poorly understood. Previously, we showed that naive Bicyclus anynana butterfly larvae, whose parents fed on a normal plant feed but coated with a novel odour, inherited an acquired preference towards that odour, which had initially elicited avoidance in the naive parents. Here, we performed simple haemolymph transfusions from odour-fed and control-fed larvae to naive larval recipients. We found that larvae injected with haemolymph from odour-fed donors stopped avoiding the novel odour, and their naive offspring preferred the odour more, compared to the offspring of larvae injected with control haemolymph. These results indicate that factors in the haemolymph, potentially the odour molecule itself, play an important role in odour learning and preference transmission across generations. Furthermore, this mechanism of odour preference inheritance, mediated by the haemolymph, bypasses the peripheral odour-sensing mechanisms taking place in the antennae, mouthparts or legs, and may mediate food plant switching and diversification in Lepidoptera or more broadly across insects.

Effects of temperature on growth, development, and survival of amphibian larvae: macroecological and evolutionary patterns.

Temperature affects the rate of biochemical and physiological processes in amphibians, influencing metamorphic traits. Temperature patterns, as those observed in latitudinal and altitudinal clines, may impose different challenges on amphibians depending on how species are geographically distributed. Moreover, species' response to environmental temperatures may also be phylogenetically constrained. Here, we explore the effects of acclimation to higher temperatures on tadpole survival, development, and growth, using a meta-analytical approach. We also evaluate whether the latitude and climatic variables at each collection site can explain differences in species' response to increasing temperature and whether these responses are phylogenetically conserved. Our results show that species that develop at relatively higher temperatures reach metamorphosis faster. Furthermore, absolute latitude at each collection site may partially explain heterogeneity in larval growth rate. Phylogenetic signal of traits in response to temperature indicates a non-random process in which related species resemble each other less than expected under Brownian motion evolution (BM) in all traits, except survival. The integration of studies in a meta-analytic framework allowed us to explore macroecological and macroevolutionary patterns and provided a better understanding of the effects of climate change on amphibians.

Buffering and phenological mismatch: A change of perspective.

The potential for climate change to disrupt phenology-mediated interactions in interaction networks has attracted considerable attention in recent decades. Frequently, studies emphasize the fragility of ephemeral seasonal interactions, and the risks posed by phenological asynchrony. Here, we argue that the fitness consequences of asynchrony in phenological interactions may often be more buffered than is typically acknowledged. We identify three main forms that buffering may take: (i) mechanisms that reduce asynchrony between consumer and resource; (ii) mechanisms that reduce the costs of being asynchronous; and (iii) mechanisms that dampen interannual variance in performance across higher organizational units. Using synchrony between the hatching of winter moth caterpillars and the leafing of their host-plants as a case study, we identify a wide variety of buffers that reduce the detrimental consequences of phenological asynchrony on caterpillar individuals, populations, and meta-populations. We follow this by drawing on examples across a breadth of taxa, and demonstrate that these buffering mechanisms may be quite general. We conclude by identifying key gaps in our knowledge of the fitness and demographic consequences of buffering, in the context of phenological mismatch. Buffering has the potential to substantially alter our understanding of the biotic impacts of future climate change-a greater recognition of the contribution of these mechanisms may reveal that many trophic interactions are surprisingly resilient, and also serve to shift research emphasis to those systems with fewer buffers and towards identifying the limits of those buffers.

Assessment of environmental and spatial factors influencing the establishment of Anopheles gambiae larval habitats in the malaria endemic province of Woleu-Ntem, northern Gabon.

BACKGROUND: This study aimed to assess the spatial distribution of Anopheles mosquito larval habitats and the environmental factors associated with them, as a prerequisite for the implementation of larviciding. METHODS: The study was conducted in December 2021, during the transition period between the end of the short rainy season (September-November) and the short dry season (December-February). Physical, biological, and land cover data were integrated with entomological observations to collect Anopheles larvae in three major towns: Mitzic, Oyem, and Bitam, using the "dipping" method during the transition from rainy to dry season. The collected larvae were then reared in a field laboratory established for the study period. After the Anopheles mosquitoes had emerged, their species were identified using appropriate morphological taxonomic keys. To determine the influence of environmental factors on the breeding of Anopheles mosquitoes, multiple-factor analysis (MFA) and a binomial generalized linear model were used. RESULTS: According to the study, only 33.1% out of the 284 larval habitats examined were found to be positive for Anopheles larvae, which were primarily identified as belonging to the Anopheles gambiae complex. The findings of the research suggested that the presence of An. gambiae complex larvae in larval habitats was associated with various significant factors such as higher urbanization, the size and type of the larval habitats (pools and puddles), co-occurrence with Culex and Aedes larvae, hot spots in ambient temperature, moderate rainfall, and land use patterns. CONCLUSIONS: The results of this research mark the initiation of a focused vector control plan that aims to eradicate or lessen the larval habitats of An. gambiae mosquitoes in Gabon's Woleu Ntem province. This approach deals with the root causes of malaria transmission through larvae and is consistent with the World Health Organization's (WHO) worldwide objective to decrease malaria prevalence in regions where it is endemic.

Selfish herd effects in aggregated caterpillars and their interaction with warning signals.

Larval Lepidoptera gain survival advantages by aggregating, especially when combined with aposematic warning signals, yet reductions in predation risk may not be experienced equally across all group members. Hamilton's selfish herd theory predicts that larvae that surround themselves with their group mates should be at lower risk of predation, and those on the periphery of aggregations experience the greatest risk, yet this has rarely been tested. Here, we expose aggregations of artificial 'caterpillar' targets to predation from free-flying, wild birds to test for marginal predation when all prey are equally accessible and for an interaction between warning coloration and marginal predation. We find that targets nearer the centre of the aggregation survived better than peripheral targets and nearby targets isolated from the group. However, there was no difference in survival between peripheral and isolated targets. We also find that grouped targets survived better than isolated targets when both are aposematic, but not when they are non-signalling. To our knowledge, our data provide the first evidence to suggest that avian predators preferentially target peripheral larvae from aggregations and that prey warning signals enhance predator avoidance of groups.

Environmental conditions shape learning in larval zebrafish.

Growing evidence reveals notable phenotypic plasticity in cognition among teleost fishes. One compelling example is the positive impact of enriched environments on learning performance. Most studies on this effect have focused on juvenile or later life stages, potentially overlooking the importance of early life plasticity. To address this gap, we investigated whether cognitive plasticity in response to environmental factors emerges during the larval stage in zebrafish. Our findings indicate that larvae exposed to an enriched environment after hatching exhibited enhanced habituation learning performance compared to their counterparts raised in a barren environment. This work underscores the presence of developmental phenotypic plasticity in cognition among teleost fish, extending its influence to the very earliest stages of an individual's life.

Tree diversity enhances predation by birds but not by arthropods across climate gradients.

Tree diversity can promote both predator abundance and diversity. However, whether this translates into increased predation and top-down control of herbivores across predator taxonomic groups and contrasting environmental conditions remains unresolved. We used a global network of tree diversity experiments (TreeDivNet) spread across three continents and three biomes to test the effects of tree species richness on predation across varying climatic conditions of temperature and precipitation. We recorded bird and arthropod predation attempts on plasticine caterpillars in monocultures and tree species mixtures. Both tree species richness and temperature increased predation by birds but not by arthropods. Furthermore, the effects of tree species richness on predation were consistent across the studied climatic gradient. Our findings provide evidence that tree diversity strengthens top-down control of insect herbivores by birds, underscoring the need to implement conservation strategies that safeguard tree diversity to sustain ecosystem services provided by natural enemies in forests.

Combining temperature-dependent life table data into Insect Life Cycle Model to forecast fall armyworm Spodoptera frugiperda (JE Smith) distribution in maize agro-ecological zones in Africa.

The fall armyworm (FAW), Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), an invasive agricultural pest, has significantly impacted crop yields across Africa. This study investigated the relationship between temperature and FAW life history traits, employing life cycle modeling at temperatures of 20, 25, 28, 30, and 32°C. The development time for eggs, larvae, and pupae varied from 0-3 days, 10-18 days, and 7-16 days, respectively. The optimal temperature range for immature stage survival and female fecundity was identified as 21-25°C, with the intrinsic rate of increase (rm) and gross reproductive rate (GRR) peaking at 25-28°C. Model validation confirmed the accuracy of these findings. The research further projected the Establishment Risk Index (ERI), Activity Index (AI), and Generation Index (GI) for FAW under current and future climates (2050 and 2070) using RCP 2.6 and RCP 8.5 scenarios. Results indicate that RCP 2.6 leads to a reduction in high-risk FAW areas, particularly in central Africa. Conversely, RCP 8.5 suggests an increase in areas conducive to FAW activity. These findings highlight the impact of climate policy on pest dynamics and the importance of incorporating climatic factors into pest management strategies. The study predicts a potential decrease in FAW prevalence in West Africa by 2070 under aggressive climate mitigation, providing a basis for future FAW management approaches.

Vegetation structure drives mosquito community composition in UK's largest managed lowland wetland.

PURPOSE: The rising burden of mosquito-borne diseases in Europe extends beyond urban areas, encompassing rural and semi-urban regions near managed and natural wetlands evidenced by recent outbreaks of Usutu and West Nile viruses. While wetland management policies focus on biodiversity and ecosystem services, few studies explore the impact on mosquito vectors. METHODS: Our research addresses this gap, examining juvenile mosquito and aquatic predator communities in 67 ditch sites within a South England coastal marsh subjected to different wetland management tiers. Using joint distribution models, we analyse how mosquito communities respond to abiotic and biotic factors influenced by wetland management. RESULTS: Of the 12 mosquito species identified, Culiseta annulata (Usutu virus vector) and Culex pipiens (Usutu and West Nile virus vector) constitute 47% of 6825 larval mosquitoes. Abundant predators include Coleoptera (water beetles) adults, Corixidae (water boatmen) and Zygoptera (Damselfy) larvae. Models reveal that tier 3 management sites (higher winter water levels, lower agricultural intensity) associated with shade and less floating vegetation are preferred by specific mosquito species. All mosquito species except Anopheles maculipennis s.l., are negatively impacted by potential predators. Culiseta annulata shows positive associations with shaded and turbid water, contrary to preferences of Corixidae predators. CONCLUSIONS: Tier 3 areas managed for biodiversity, characterised by higher seasonal water levels and reduced livestock grazing intensity, provide favourable habitats for key mosquito species that are known vectors of arboviruses, such as Usutu and West Nile. Our findings emphasise the impact of biodiversity-focused wetland management, altering mosquito breeding site vegetation to enhance vector suitability. Further exploration of these trade-offs is crucial for comprehending the broader implications of wetland management.

Sleep pressure modulates single-neuron synapse number in zebrafish.

Sleep is a nearly universal behaviour with unclear functions1. The synaptic homeostasis hypothesis proposes that sleep is required to renormalize the increases in synaptic number and strength that occur during wakefulness2. Some studies examining either large neuronal populations3 or small patches of dendrites4 have found evidence consistent with the synaptic homeostasis hypothesis, but whether sleep merely functions as a permissive state or actively promotes synaptic downregulation at the scale of whole neurons is unclear. Here, by repeatedly imaging all excitatory synapses on single neurons across sleep-wake states of zebrafish larvae, we show that synapses are gained during periods of wake (either spontaneous or forced) and lost during sleep in a neuron-subtype-dependent manner. However, synapse loss is greatest during sleep associated with high sleep pressure after prolonged wakefulness, and lowest in the latter half of an undisrupted night. Conversely, sleep induced pharmacologically during periods of low sleep pressure is insufficient to trigger synapse loss unless adenosine levels are boosted while noradrenergic tone is inhibited. We conclude that sleep-dependent synapse loss is regulated by sleep pressure at the level of the single neuron and that not all sleep periods are equally capable of fulfilling the functions of synaptic homeostasis.

Telomere length is longer following diapause in two solitary bee species.

The mechanisms that underlie senescence are not well understood in insects. Telomeres are conserved repetitive sequences at chromosome ends that protect DNA during replication. In many vertebrates, telomeres shorten during cell division and in response to stress and are often used as a cellular marker of senescence. However, little is known about telomere dynamics across the lifespan in invertebrates. We measured telomere length in larvae, prepupae, pupae, and adults of two species of solitary bees, Osmia lignaria and Megachile rotundata. Contrary to our predictions, telomere length was longer in later developmental stages in both O. lignaria and M. rotundata. Longer telomeres occurred after emergence from diapause, which is a physiological state with increased tolerance to stress. In O. lignaria, telomeres were longer in adults when they emerged following diapause. In M. rotundata, telomeres were longer in the pupal stage and subsequent adult stage, which occurs after prepupal diapause. In both species, telomere length did not change during the 8 months of diapause. Telomere length did not differ by mass similarly across species or sex. We also did not see a difference in telomere length after adult O. lignaria were exposed to a nutritional stress, nor did length change during their adult lifespan. Taken together, these results suggest that telomere dynamics in solitary bees differ from what is commonly reported in vertebrates and suggest that insect diapause may influence telomere dynamics.

Seasonal fish larvae abundance and composition in seagrass habitats of coastal East Africa.

Seagrass habitats play a major role in fisheries productivity through nursery functions and feeding grounds for diverse fish species. However, little is known about the seasonal distribution of fish larvae at large spatial scales in coastal East Africa. We investigated drivers of the seasonal fish larvae abundance and composition in seagrass habitats in Kenya and Tanzania. We found a high diversity of fish larvae (54 families) inhabiting seagrass habitats that differed between sites and seasons. Fish larvae abundance were highest in Kenya, particularly during the northeast monsoon season. Overall, total larval abundances per site were low, reaching less than 190 individuals/100 m3 in Kenya and less than 40 individuals/100 m3 in Tanzania, likely related to the low productivity and strong hydrodynamic processes in this region. Our data suggests that most of the fish spawn year-round in these tropical waters as we did not find strong seasonal patterns. All sites had a high relative abundance of larvae from demersal spawning fishes, indicating that many fish species move to coastal sites for spawning. Primary productivity and dissolved oxygen, driven by hydrodynamics conditions are positively related to fish larvae productivity both in Kenya and Tanzania. These findings indicate that the occurrence of both resident and transient fish larvae in seagrass meadows is driven by strong hydrodynamic and tidal processes that transport fish larvae across adjacent habitats.

Release of Angiostrongylus cantonensis larvae from live intermediate hosts under stress.

The metastrongyloid nematode Angiostrongylus cantonensis causes eosinophilic meningitis in a variety of homeothermic hosts including humans. Third-stage infectious larvae develop in gastropods as intermediate hosts. Humans are usually infected by intentional or incidental ingestion of an infected mollusk or paratenic host (poikilothermic vertebrates and invertebrates). The infection may also hypothetically occur through ingestion of food or water contaminated by third-stage larvae spontaneously released from gastropods. Larvae are thought to be released in greater numbers from the intermediate host exposed to stress. This study aimed to compare larval release from stressed with unstressed gastropods. Experimentally infected Limax maximus and Lissachatina fulica were exposed to a stress stimulus (shaking on an orbital shaker). The mucus was collected before and after the stress and examined microscopically and by qPCR for the presence of A. cantonensis larvae and their DNA. In the case of L. maximus, no larvae were detected microscopically in the mucus, but qPCR analysis confirmed the presence of A. cantonensis DNA in all experimental replicates, without clear differences between stressed and non-stressed individuals. In contrast, individual larvae of A. cantonensis were found in mucus from Li. fulica after stress exposure, which also reflects an increased number of DNA-positive mucus samples after stress. Stress stimuli of intensity similar to the transport or handling of mollusks can stimulate the release of larvae from highly infected intermediate hosts. However, these larvae are released in small numbers. The exact number of larvae required to trigger neuroangiostrongyliasis is unknown. Therefore, caution is essential when interacting with potential intermediate hosts in regions where A. cantonensis is endemic.

Predation cues induce predator specific changes in olfactory neurons encoding defensive responses in agile frog tadpoles.

Although behavioural defensive responses have been recorded several times in both laboratory and natural habitats, their neural mechanisms have seldom been investigated. To explore how chemical, water-borne cues are conveyed to the forebrain and instruct behavioural responses in anuran larvae, we conditioned newly hatched agile frog tadpoles using predator olfactory cues, specifically either native odonate larvae or alien crayfish kairomones. We expected chronic treatments to influence the basal neuronal activity of the tadpoles' mitral cells and alter their sensory neuronal connections, thereby impacting information processing. Subsequently, these neurons were acutely perfused, and their responses were compared with the defensive behaviour of tadpoles previously conditioned and exposed to the same cues. Tadpoles conditioned with odonate cues differed in both passive and active cell properties compared to those exposed to water (controls) or crayfish cues. The observed upregulation of membrane conductance and increase in both the number of active synapses and receptor density at the postsynaptic site are believed to have enhanced their responsiveness to external stimuli. Odonate cues also affected the resting membrane potential and firing rate of mitral cells during electrophysiological patch-clamp recordings, suggesting a rearrangement of the repertoire of voltage-dependent conductances expressed in cell membranes. These recorded neural changes may modulate the induction of an action potential and transmission of information. Furthermore, the recording of neural activity indicated that the lack of defensive responses towards non-native predators is due to the non-recognition of their olfactory cues.

Wavelength-specific negatively phototactic responses of the burrowing mayfly larvae Ephoron virgo.

Mayflies are typically negatively phototactic during larval development, whereas the adults possess positive phototaxis. However, no extensive research has been done into the wavelength dependence of phototaxis in any mayfly larvae. We measured the repellency rate of Ephoron virgo larvae to light as a function of wavelength in the 368-743 nm spectral range. We established that the magnitude of repellence increased with decreasing wavelength and the maximal responses were elicited by 400 nm violet light. This wavelength dependence of phototaxis is similar to the recently reported spectral sensitivity of positive phototaxis of the twilight-swarming E. virgo adults. Negative phototaxis not only facilitates predation evasion: avoidance of the blue-violet spectral range could also promote the larvae to withdraw towards the river midline in the case of a drop in the water level, when the underwater light becomes enriched with shorter wavelengths as a result of the decreasing depth of overhead river water.

Bacterial biota associated with the invasive insect pest Tuta absoluta (Meyrick).

Tuta absoluta (the tomato pinworm) is an invasive insect pest with a highly damaging effect on tomatoes causing between 80 and 100% yield losses if left uncontrolled. Resistance to chemical pesticides have been reported in some T. absoluta populations. Insect microbiome plays an important role in the behavior, physiology, and survivability of their host. In a bid to explore and develop an alternative control method, the associated microbiome of this insect was studied. In this study, we unraveled the bacterial biota of T. absoluta larvae and adults by sequencing and analyzing the 16S rRNA V3-V4 gene regions using Illumina NovaSeq PE250. Out of 2,092,015 amplicon sequence variants (ASVs) recovered from 30 samples (15 larvae and 15 adults), 1,268,810 and 823,205 ASVs were obtained from the larvae and adults, respectively. A total of 433 bacterial genera were shared between the adults and larval samples while 264 and 139 genera were unique to the larvae and adults, respectively. Amplicon metagenomic analyses of the sequences showed the dominance of the phylum Proteobacteria in the adult samples while Firmicutes and Proteobacteria dominated in the larval samples. Linear discriminant analysis effect size (LEfSe) comparison revealed the genera Pseudomonas, Delftia and Ralstonia to be differentially enriched in the adult samples while Enterococcus, Enterobacter, Lactococcus, Klebsiella and Wiessella were differentially abundant in the larvae. The diversity indices showed that the bacterial communities were not different between the insect samples collected from different geographical regions. However, the bacterial communities significantly differed based on the sample type between larvae and adults. A co-occurrence network of significantly correlated taxa revealed a strong interaction between the microbial communities. The functional analysis of the microbiome using FAPROTAX showed that denitrification, arsenite oxidation, methylotrophy and methanotrophy as the active functional groups of the adult and larvae microbiomes. Our results have revealed the core taxonomic, functional, and interacting microbiota of T. absoluta and these indicate that the larvae and adults harbor a similar but transitory set of bacteria. The results provide a novel insight and a basis for exploring microbiome-based biocontrol strategy for this invasive insect pest as well as the ecological significance of some of the identified microbiota is discussed.

Extracellular traps development in canine neutrophils induced by infective stage Toxocara canis larvae.

Neutrophils, a crucial element of the host defense system, develop extracellular traps against helminth parasites. Neutrophils accumulate around the larvae of Toxocara canis (T. canis) in the tissues of the organism. This study aimed to determine the reaction in canine neutrophils after incubation with infective stage T. canis larvae (L3) in vitro. Most L3 were still active and moved between the extracellular traps (NETs) after 60-min incubation. NETs were not disintegrated by L3 movement. The L3 was only immobilized by NETs, entrapped larvae were still motile between the traps at the 24 h incubation. NETs were observed not only to accumulate around the mouth, excretory pole or anus but also the entire body of live L3. The extracellular DNA amount released from the canine neutrophils after being induced with phorbol 12-myristate 13-acetate was not affected by T. canis excretory/secretory products obtained from 250 L3. To the Authors'knowledge, the extracellular trap structures was firstly observed in canine neutrophils against T. canis L3 in vitro. NETs decorated with myeloperoxidase, neutrophil elastase and histone (H3) were observed under fluorescence microscope. There were not significant differences in the amount of extracellular DNA (P > 0.05), but the morphological structure of NETs was different in the live and head-inactivated T. canis larvae.

Girdling behavior of the longhorn beetle modulates the host plant to enhance larval performance.

BACKGROUND: Preingestive behavioral modulations of herbivorous insects on the host plant are abundant over insect taxa. Those behaviors are suspected to have functions such as deactivation of host plant defenses, nutrient accumulation, or modulating plant-mediated herbivore interactions. To understand the functional consequence of behavioral modulation of insect herbivore, we studied the girdling behavior of Phytoecia rufiventris Gautier (Lamiinae; Cerambycidae) on its host plant Erigeron annuus L. (Asteraceae) that is performed before endophytic oviposition in the stem. RESULTS: The girdling behavior significantly increased the larval performance in both field monitoring and lab experiment. The upper part of the girdled stem exhibited lack of jasmonic acid induction upon larval attack, lowered protease inhibitor activity, and accumulated sugars and amino acids in compared to non-girdled stem. The girdling behavior had no effect on the larval performance of a non-girdling longhorn beetle Agapanthia amurensis, which also feeds on the stem of E. annuus during larval phase. However, the girdling behavior decreased the preference of A. amurensis females for oviposition, which enabled P. rufiventris larvae to avoid competition with A. amurensis larvae. CONCLUSIONS: In conclusion, the girdling behavior modulates plant physiology and morphology to provide a modulated food source for larva and hide it from the competitor. Our study implies that the insect behavior modulations can have multiple functions, providing insights into adaptation of insect behavior in context of plant-herbivore interaction.

Reduced ossification caused by 3D simulated microgravity exposure is short-term in larval zebrafish.

Understanding how skeletal tissues respond to microgravity is ever more important with the increased interest in human space travel. Here, we exposed larval Danio rerio at 3.5 dpf to simulated microgravity (SMG) using a 3D mode of rotation in a ground-based experiment and then studied different cellular, molecular, and morphological bone responses both immediately after exposure and one week later. Our results indicate an overall decrease in ossification in several developing skeletal elements immediately after SMG exposure with the exception of the otoliths, however ossification returns to normal levels seven days after exposure. Coincident with the reduction in overall ossification tnfsf11 (RANKL) expression is highly elevated after 24 h of SMG exposure and also returns to normal levels seven days after exposure. We also show that genes associated with osteoblasts are unaffected immediately after SMG exposure. Thus, the observed reduction in ossification is primarily the result of a high level of bone resorption. This study sheds insight into the nuances of how osteoblasts and osteoclasts in the skeleton of a vertebrate organism respond to an external environmental disturbance, in this case simulated microgravity.

Bacterial c-di-GMP signaling gene affects mussel larval metamorphosis through outer membrane vesicles and lipopolysaccharides.

Biofilms serve as crucial cues for settlement and metamorphosis in marine invertebrates. Within bacterial systems, c-di-GMP functions as a pivotal signaling molecule regulating both biofilm formation and dispersion. However, the molecular mechanism of how c-di-GMP modulates biofilm-induced larval metamorphosis remains elusive. Our study reveals that the deletion of a c-di-GMP related gene in Pseudoalteromonas marina led to an increase in the level of bacterial c-di-GMP by knockout technique, and the mutant strain had an enhanced ability to produce more outer membrane vesicles (OMVs) and lipopolysaccharides (LPS). The mutant biofilms had higher induction activity for larval metamorphosis in mussels Mytilus coruscus, and OMVs play a major role in the induction activity. We further explored the function of LPS in OMVs. Extracted LPS induced high larval metamorphosis rate, and LPS content were subject to c-di-GMP and LPS-biosynthesis gene. Thus, we postulate that the impact of c-di-GMP on biofilm-induced metamorphosis is mediated through OMVs and LPS.