Larvae Research Articles

Recently Evolved, Stage-Specific Genes Are Enriched at Life-Stage Transitions in Flies.

Understanding how genomic information is selectively utilized across different life stages is essential for deciphering the developmental and evolutionary strategies of metazoans. In holometabolous insects, the dynamic expression of genes enables distinct functional adaptations at embryonic, larval, pupal, and adult stages, likely contributing to their evolutionary success. While Drosophila melanogaster (D. melanogaster) has been extensively studied, less is known about the evolutionary dynamics that could govern stage-specific gene expression. To address this question, we compared the distribution of stage-specific genes, that is, genes expressed in temporally restricted developmental stages, across the development of D. melanogaster and Aedes aegypti (A. aegypti). Using tau-scoring, a computational method to determine gene expression specificity, we found that, on average, a large proportion of genes (20%-30% of all protein-coding genes) in both species exhibit restricted expression to specific developmental stages. Phylostratigraphy analysis, a method to date the age of genes, further revealed that stage-specific genes fall into two major categories: highly conserved and recently evolved. Notably, many of the recently evolved and stage-specific genes identified in A. aegypti and D. melanogaster are restricted to Diptera order (20%-35% of all stage-specific genes), highlighting ongoing evolutionary processes that continue to shape life-stage transitions. Overall, our findings underscore the complex interplay between gene evolutionary age, expression specificity, and morphological transformations in development. These results suggest that the attraction of genes to critical life-stage transitions is an ongoing process that may not be constant across evolutionary time or uniform between different lineages, offering new insights into the adaptability and diversification of dipteran genomes.

Fusarium proliferatum: isolation, identification and evaluation of its association with the model insect Galleria mellonella (L.) (Lepidoptera: Pyralidae)

Although the genus Fusarium is widely recognized for including significant plant pathogens and mycotoxin producers, many Fusarium species have also demonstrated promising potential for insect control. These species exhibit characteristics such as high mortality rates, rapid action, and prolific sporulation, making them attractive candidates for agricultural pest management. In this study, the micro and macromorphology, DNA sequencing, and the effects on the model insect Galleria mellonella of Fusarium proliferatum isolated from soil using the trap method were investigated. Morphological analyses revealed detailed colony and spore structures on various agar media. Species identification was confirmed through DNA analysis using ITS and CaM gene sequences. F. proliferatum was observed to prolong the larval stage of model ınsect G. mellonella while reducing pupal duration, weight, and total egg production. Our findings indicate that the fungus exerts a dose-dependent impact on the development and reproduction of G. mellonella. Future studies should focus on identifying secondary metabolites responsible for the observed biological effects, evaluating possible effects on non-target organisms, and conducting field trials under different environmental conditions.

First Report of the Anti-Parasitic Effect of a Cannabis sativa full-spectrum Extract on Echinococcus granulosus sensu stricto.

Cystic echinococcosis is a parasitic zoonosis caused by the larval stage of Echinococcus granulosus sensu lato. Albendazole (ABZ) is the drug of choice, although its efficacy is variable. The present research aimed to assess the in vitro and in vivo efficacy of a full-spectrum extract of Cannabis sativa inflorescences against E. granulosus sensu stricto (s.s.). Protoscoleces and cysts were incubated in vitro with the C. sativa extract, achieving final CBD concentrations of 1, 5, 10, and 50µg/ml. Viability was evaluated periodically. Structural and ultrastructural alterations were also recorded. For the clinical efficacy study, female CF-1 mice were infected. Six months later, mice were divided into groups (n = 10): (a) water control; (b) ABZ; (c) C. sativa extract, and (d) ABZ + C. sativa extract. Treatments were administered every 24h for 30 days. The efficacy of the treatments was evaluated according to the weight of the cysts collected and the ultrastructural alterations observed. The C. sativa extract caused a significant decrease in the viability of protoscoleces and cysts in vitro. The greatest effect was observed with 50µg/ml, which generated the reduction in protoscoleces viability to 0% between 6 and 24h post-incubation (pi) and the collapse of 92 ± 13% of the cysts after 24h pi. All the in vivo treatments reduced the weight of the cysts and caused ultrastructural alterations, especially the combination of ABZ + C. sativa extract. We demonstrated the in vitro and in vivo efficacy of a full-spectrum extract of C. sativa inflorescences against E. granulosus s.s.

Spatiotemporal dynamics of histone H3K27 demethylase during development.

Spatiotemporal dynamics of histone H3K27 demethylase during development.

Performance of the Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), over Three Generations on Four Maize Cultivars

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is a highly destructive pest that poses serious threats and causes significant losses to the production of maize in China. This study evaluated the feeding and oviposition preferences of S. frugiperda when reared on four maize cultivars—sweet, waxy, common, and silage—across three consecutive generations. It also compared population adaptability among these cultivars and analyzed population parameters between the F1 and F3 generations. The findings revealed that all four F1 generation populations showed a preference for feeding and oviposition on sweet maize. However, over time, S. frugiperda exhibited a stronger preference, in terms of feeding and oviposition behaviors, for the natal host plant across three consecutive generations of rearing. The fall armyworm completed its life cycle and oviposited on all four maize varieties over three generations. The sweet cultivar population had the highest intrinsic rate of increase, finite rate of increase, net reproductive rate, larval survival rate, pupation rate, eclosion rate, fecundity, and pupal weight, while the silage cultivar population had the shortest larval stage, pre-adult stage, and adult lifespan and the pupal weight and the fecundity were the lowest. Overall, the population fitness was the highest on the sweet cultivar, and the lowest on the silage cultivar. Compared with F1, the F3 generation of the FAW had a significantly shorter developmental duration in four maize cultivars. Except for the waxy maize cultivars, the fecundity of the other three cultivars did not differ significantly between F1 and F3. This study provides fundamental information on the trend of fall armyworm population changes in maize fields and serves as a reference for rational maize cultivar planting decisions.

A Study on the Life Table of the Fruit Borer (Helicoverpa armigera) on Chilli

The overall duration necessary for the life cycle to progress from eggs to the emergence of adults ranged from 33 to 44 days, with an average of 38.5 ± 7.78 days. In contrast, the complete life cycle from eggs to the death of both male and female was observed to be finalized within 39 to 52 days, averaging 45.5 ± 9.19 days for males and 40 to 53 days, averaging 46.5 ± 9.19 days for females. Specifically, the time required for egg hatching was approximately 3 to 4 days, with an average of 3.5 days. The total larval stage lasted about 17 to 23 days, averaging 20 days, while the pre-pupal phase took 1 to 2 days, averaging 1.5 days. The pupal stage lasted around 12 to 15 days, with an average of 13.5 days. The pre-oviposition period extended from 2 to 5 days, averaging 3.5 days, and the oviposition period lasted 5 to 7 days, with an average of 6 days. The post-oviposition period was about 1 to 2 days, averaging 1.5 days. The longevity of adult males and females varied slightly depending on food availability. Males with food lived for approximately 6 to 8 days, averaging 7 days, while females survived for about 7 to 9 days. Males without food had a lifespan of about 1 to 2 days, averaging 1.5 days, whereas females lived for about 1 to 3 days, with an average of around 2 days. The total life cycle duration for male moths with food was between 39 to 52 days, averaging 45.5 days, while the total life cycle for female moths with food ranged from 40 to 53 days, averaging 46.5 days.

The impact of the BmSur8 gene, encoding a scaffold protein, on cocoon silk yield in Bombyx mori.

Cocoon silk quality is a critical determinant of the economic value in the sericulture industry for Bombyx mori L. (Lepidoptera: Bombycidae). Sur8 functions as a positive regulator of the RAS-MAPK signaling cascade, which plays an essential role in regulating cocoon silk production in Bombyx mori. However, the specific functions of BmSur8 in silkworms have not been fully elucidated. In this study, we demonstrate that BmSur8 is a highly conserved gene expressed across all life stages and various tissues at the third day of the fifth instar larvae (L5D3) stage in Bombyx mori. Knockout of the BmSur8 gene results in a 15.2% reduction in silk yield in females and a 13.4% reduction in males, with the cocoon layer rate in females decreasing by 3.8%. Through transcriptomic analysis of posterior silk glands (PSGs) at L5D3 stage from ΔBmSur8 mutants and Nistari controls, as well as mass spectrometry analysis of BmSur8-interacting proteins in BmN cells, we find that BmSur8 potentially interacts with BmEtfb and regulates the expression of BmCDA and BmjunD, thereby participating in material and energy metabolism. This study provides a comprehensive analysis of the function of BmSur8 in metabolic pathways and offers theoretical insights and experimental data for researching silkworm cocoon silk quality and yield, as well as for breed selection and breeding.

Developmental transcriptomics reveals stage-specific immune gene expression profiles in Spodoptera frugiperda

The fall armyworm, Spodoptera frugiperda, a globally invasive pest, demonstrates distinct immune adaptations across developmental stages and sexes, which are critical for its survival and adaptability. Using high-throughput RNA sequencing, this study systematically profiled 56 immune-related gene families, identifying 157 genes involved in Toll and Imd signaling pathways, and 185 genes associated with cellular immunity. Dynamic expression patterns were observed, with humoral immunity indices peaking during the third (L3) and fifth (L5) instars and diminishing in the pupal (P) and egg stages. In contrast, cellular immunity indices were highest in pupae and adult females, while the sixth instar (L6) and adult males exhibited the lowest immune capacity. Female adults displayed superior immune potential compared to males, reflecting evolutionary pressures tied to reproductive fitness. Notably, larval stages exhibited heightened immune gene expression, which aligns with their vulnerability to pathogens. Validation via qRT-PCR confirmed these transcriptomic trends, highlighting the modulation of immunity throughout development. These findings offer novel insights into the interplay between developmental progression and immune regulation in S. frugiperda. By elucidating these stage-specific immune responses, this study provides a robust framework for developing targeted pest management strategies aimed at mitigating the impact of this destructive pest.

Stage-dependent life-history, physiological, and behavioral responses to low pH in an estuarine crab.

Stage-dependent life-history, physiological, and behavioral responses to low pH in an estuarine crab.

Genetic and behavioral analyses suggest that larval and adult stages of Lucilia cuprina employ different sensory systems to detect rotten beef

BackgroundThe blowfly Lucilia cuprina is a destructive parasite of sheep that causes flystrike or myiasis. Larvae consume the animal’s living flesh, producing large wounds that can lead to death. The main aim of this study was to identify genes that may play important roles in the behavior and physiology of L. cuprina larvae.MethodsAn RNA-Seq analysis of RNA from whole larvae at different developmental stages and third-instar head and gut tissues was used to identify sensory receptors and other genes relevant to the physiology of L. cuprina larvae. In addition, CRISPR/Cas9 gene editing was used to obtain a loss-of-function mutation for the L. cuprina odorant coreceptor gene (LcupOrco). The response of mutant larvae and adult females to fresh and rotten meat at different temperatures was evaluated.ResultsThe RNA-Seq analysis suggested that odorant (OR), gustatory, ionotropic, and Pickpocket receptors may not play a central role in the L. cuprina larval sensory signaling and digestive systems. Rather, ATP-binding cassettes (ABCs) were highly enriched in head and gut RNA, and odorant-binding proteins (OBPs) only in the head. To confirm that ORs are not essential for larval detection of rotten beef, diet-choice assays were performed including larvae and adults homozygous for a null mutation in LcupOrco. While the attraction of adult females to rotten beef was disrupted, LcupOrco mutant larvae showed no change in diet preference.ConclusionsThe expression pattern of the ABC and OBP gene families suggests a central role in the sensory system of the L. cuprina larva for these receptors. Behavioral assays showed that ORs are essential for the adult female response to rotten beef, but not for larval behavior. These findings are consistent with high levels of expression of LcupOrco in the adult female antenna but very low expression in larvae.Graphical abstract

Hypoxia to the skeletal systems of teleost: Impacts, mechanisms, and health implications.

Hypoxia to the skeletal systems of teleost: Impacts, mechanisms, and health implications.

Butylated Hydroxyanisole (BHA) Disrupts Brain Signalling in Embryo–Larval Stage of Zebrafish Leading to Attention Deficit Hyperactivity Disorder (ADHD)

Background: Butylated hydroxyanisole (BHA) has been extensively used in several commercial industries as a preservative. It causes severe cellular and neurological damage affecting the developing fetus and might induce attention deficit hyperactivity disorder (ADHD). Methods: Zebrafish embryos were subjected to five distinct doses of BHA—0.5, 1, 2, 4, and 8 ppb up to 96 h post fertilization (hpf). Hatching rate, heart rate, and body malformations were assessed at 48 hpf, 72 hpf, and 48–96 hpf, respectively. After exposure, apoptotic activity, neurobehavioral evaluation, neurotransmitter assay, and antioxidant activity were assessed at 96 hpf. At 120 hpf, the expression of genes DRD4, COMT, 5-HTR1aa, and BDNF was evaluated by real-time PCR. Results: BHA exposure showed a delay in the hatching rate and a decrease in the heart rate of the embryo when compared with the control. Larvae exhibited developmental deformities such as bent spine, yolk sac, and pericardial edema. A higher density of apoptotic cells was observed in BHA-exposed larvae at 96 hpf. There was a decline in catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and superoxide dismutase (SOD) activity, indicating oxidative stress. There was a significant decrease in Acetylcholinesterase (AChE) activity and serotonin levels with an increase in concentration of BHA, leading to a dose-responsive increase in anxiety and impairment in memory. A significant decrease in gene expression was also observed for DRD4, COMT, 5-HTR1aa, and BDNF. Conclusions: Even at lower concentrations of BHA, zebrafish embryos suffered from developmental toxicity, anxiety, and impaired memory due to a decrease in AChE activity and serotonin levels and altered the expression of the mentioned genes.

Effects of Photoperiod on the Developmental Duration and Reproduction of Sclerodermus sichuanensis

Sclerodermus sichuanensis, a parasitic wasp, plays a significant role in wood-boring forest pest control in China. Research has shown that the photoperiod significantly affects the development and reproduction of parasitic wasps. However, the effects of the photoperiod on S. sichuanensis have not yet been reported. This study investigates the impact of different photoperiods on the developmental duration and reproduction of S. sichuanensis. The wasps were reared under four photoperiod conditions: 0L:24D, 8L:16D, 16L:8D, and 24L:0D. The results show that increased light duration shortened the egg, larval, and pupal stages, with the total developmental period decreasing by up to 17 days. The number of offspring was highest in the 8L:16D and 16L:8D treatments, while constant light (24L:0D) led to a significant decrease in offspring numbers. The parasitism rate and pre-oviposition period were also affected by light exposure, with the longest pre-oviposition period observed in complete darkness. These findings indicate that the photoperiod plays a crucial role in regulating the development and reproductive efficiency of S. sichuanensis, suggesting that appropriate light conditions could enhance the efficiency of its use in pest control.

Comparative susceptibility of wild and laboratory-reared Aedes and Anopheles larvae to ivermectin

BackgroundAdministering ivermectin to humans and livestock renders their blood toxic for mosquitoes like Anopheles and Aedes, offering a promising approach for controlling these vectors. However, the impact of such treatment on larval stages exposed to the drug through contaminated breeding sites is not fully understood. This study looked at how ivermectin affects the development of Aedes and Anopheles larvae.MethodsFour instars laboratory-reared (Anopheles gambiae Kisumu strain and Aedes aegypti Bora Bora strain) and wild-derived (Anopheles coluzzii VK5 and Ae. aegypti Bobo) larvae were exposed to ivermectin-medium containing the molecule at concentrations ranging from 0 to 100 ng/ml for 24 h, then transferred surviving larvae into ivermectin-free medium to monitor development until adult stage and female fecundity. Parameters measured were: larval survival, pupation dynamics, teneral emergence rates, and fecundity of the adult females in terms of numbers of eggs developed and laid. Two independent experiments were performed, each with four biological replicates. Data obtained for each life history parameter were compared between treatments to characterize ivermectin effects.ResultsData indicated that highest ivermectin concentrations (100, 75, and 50 ng/ml) reduced larval survival by over 50% within 24 to 48 h post-exposure, with varying effects across different strains. Wild-derived larvae showed lower susceptibility to ivermectin compared to laboratory larvae for both Anopheles and Aedes species. The concentrations leading to 50% larval mortality (4-day-LC50) were 3.65 and 1.86 ng/ml for Anopheles VK5 and Kisumu strains, and 15.60 and 2.56 ng/ml for Aedes Bobo and Bora Bora strains, respectively. Notably, while high concentrations severely impacted larval development, low concentration (1 ng/ml) appear to be a sublethal concentration and allowed for adult emergence. No significant effects on the number of laid eggs were observed across the different strains.ConclusionOverall, these data showed how development parameters of laboratory-raised and wild-derived Anopheles and Aedes larvae are affected differently by ivermectin, highlighting potential implications for vector control strategies and ecological concerns regarding non-target organisms and environment persistence. Further investigations are planned to understand existing mechanisms allowing wild-derived larvae to better survive than laboratory ones despite the presence of ivermectin in their breeding environment.

Comparative immunomodulatory effects of chronic exposure to imidacloprid and thiamethoxam on the respiratory burst response in zebrafish and fathead minnow larvae.

Comparative immunomodulatory effects of chronic exposure to imidacloprid and thiamethoxam on the respiratory burst response in zebrafish and fathead minnow larvae.

Effects of long-term thermal stress on population dynamics and HSP70 expression of Hyphantria cunea (Lepidoptera: Erebidae).

Hyphantria cunea (Lepidoptera: Erebidae), a globally significant quarantine pest, is facing thermal stress during its southward expansion in China. Heat shock protein 70 (HSP70) family members serve as key components in insect molecular networks under heat stress. However, studies remain limited on this pest's tolerance to persistent high temperatures and the role of HSP70 under such stress conditions. In this study, extreme heat treatment (EHT; daily cycle of 31 °C (6h), 34 °C (6h), 37 °C (6h), and 34 °C (6h)) drastically reduced H. cunea larval survival, preventing life cycle completion. Heat treatment (HT; daily cycle of 28 °C (6h), 31 °C (6h), 34 °C (6h), and 31 °C (6h)) during early stages had minor effects on larval development, whereas exposure during late larval stages shortened developmental duration, reduced fecundity, and decreased fitness. To explore the molecular mechanisms, four HSP70 genes of H. cunea were cloned and characterized. Phylogenetic analysis revealed high conservation among HcHSP70s, showing a close relationship with those from Noctuidae insects. HcHSP70 transcripts were significantly upregulated during heat stress, exhibiting diurnal fluctuation with peak expression at specific times. These results show that H. cunea can tolerate moderate heat stress, but pre-reproductive thermal stress reduces fecundity. The upregulation of HSP70 expression contributes to enhanced thermotolerance in this pest. Overall, this research provides a reference for population prediction of H. cunea in regions with increasing heat stress.

Corn Oil Supplementation Enhances Locomotor Performance and Mitochondrial Function in Drosophila melanogaster

Polyunsaturated fatty acids are crucial for brain health, aiding cognitive development and helping to prevent neurodegenerative diseases. Since the body cannot produce them, it is essential to obtain them from food. This study aimed to analyze the effect of corn oil on the behavior and biochemical parameters of Drosophila melanogaster. D. melanogaster were fed a diet supplemented with corn oil at varying concentrations from the larval stage until the fifth day of adulthood. A corn oil diet (37.8 mg/mL of linoleic acid) decreased mortality under starvation conditions and enhanced locomotor performance (p < 0.01). Biochemical analyses revealed decreased levels of glutathione (p < 0.001), citrate synthase activity (p < 0.05), and mitochondrial phosphorylation (p < 0.05), indicating a potential boost in energy metabolism. Conversely, a decrease in acetylcholinesterase (p < 0.05) was noted, suggesting cholinergic modulation. These findings demonstrate that corn oil supplementation benefits neural health in this animal model, paving the way for further exploration of non-pharmacological treatments for neurodegenerative processes such as Alzheimer’s disease. Biochemical analyses showed decreased levels of glutathione (p < 0.001), citrate synthase activity (p < 0.05), and mitochondrial phosphorylation (p < 0.05), suggesting a possible increase in energy metabolism.

Water temperature modulates multidimensional plastic responses to water flow during the ontogeny of a neotropical fish (Astyanax lacustris, characiformes)

IntroductionPlastic phenotypes result from multidimensional developmental systems responding to distinct yet simultaneous environmental signals, which may differently affect the magnitude and directions of plastic responses.Concomitant environmental signals during development may result in dominant, synergistic, or even antagonistic phenotypic effects, so that a given condition may amplify or minimize plastic responses to other environmental stimuli. Knowledge on how external information shapes complex plastic phenotypes is essential to predict potential evolutionary trajectories driven by developmental plasticity.MethodsHere, we manipulate water temperature to evaluate its effects on the well-described phenotypic accommodation of fish growth in the presence of water flow, using the neotropical species Astyanax lacustris. We include larval and juvenile ontogenetic stages to examine the interaction between these two environmental signals in plastic responses related to body size and shape, skeleton ossification and gene expression, using bmp4 as a proxy for ossification pathways.Results and discussionOur results demonstrate that water temperature plays a crucial role determining the expression of plastic variation at all dimensions, and effects of water flow were restricted to specific thermal regimes. Combination of high temperature and water flow has a major effect on body shape and unveils unique phenotypic patterns, supporting the prediction that high temperatures can amplify plastic responses to external signals. Specifically, fish raised in the presence of water flow at warmer environments grew faster and ossified earlier, and this condition increased bmp4 expression levels especially at later developmental stages. Such plastic phenotypes likely involve a functional relationship with swimming performance in running-water environments. Our findings highlight the importance of studying developmental plasticity in complex environments using a multidimensional approach, especially considering increments in water temperatures due to accelerated climate changes that likely impact the fish developmental potential to mitigate environmental changes through plastic responses.

Chemical profiling of developmental stages in three major flies via direct infusion mass spectrometry: Improving estimates of minimum postmortem intervals in forensic entomology.

Forensic entomology has become an essential tool for estimating the minimum postmortem interval (mPMI), especially in scenarios where traditional methods are limited. This study aimed to characterize and differentiate the chemical profiles of three major forensically relevant fly species-Chrysomya albiceps, Cochliomyia macellaria, and Lucilia cuprina-across their developmental stages. Changes in chemical profiles during larval, pupal, and pharate adult stages were monitored via ethanol-based solvent extraction followed by direct infusion mass spectrometry (DIMS). Distinct chemical fingerprints were observed for each stage, with specific variations in ion compositions reflecting the dynamic nature of insect development. Chemometric analysis using PLS-DA models clearly distinguished the species and their developmental stages, especially during the larval phase, which is critical for mPMI estimation. These findings demonstrate that metabolomic profiling combined with chemometric tools offers, in forensic investigations, a precise and robust approach to estimate mPMI and to identify fly species in their larval stages.

Phylogeny and reclassification of Limacodidae worldwide, with emphasis on the ontogeny of larval defenses (Lepidoptera: Zygaenoidea)

Abstract Limacodidae (Zygaenoidea), a mostly pantropical family of 1,800+ species, is known for sluglike caterpillars with suckers below rather than typical prolegs. They typically have either venomous spines (nettles) or a smooth surface (gelatines) above. Previous limacodid phylogenies were of limited geographic scope or narrow taxonomic focus, thus unable to show the family’s internal relationships comprehensively. Our phylogenetic hypotheses include 95 genera and 125 species of Limacodidae (stat. nov.) of worldwide distribution, doubling genera, and tripling species numbers represented from previous studies. Additionally, 14 species in other zygaenoid families and 6 outside Zygaenoidea are included. In contrast to other works based on independent analysis of either molecular or morphological data, our limacodid-focused trees are the first ones to combine molecular data with morphological characters from adults and immature stages. Resulting topologies are based on 1 mitochondrial and 6 nuclear genes, totaling 5,153 base pairs, and 122 morphological characters (5,275 characters in total). Our trees support the monophyly of Zygaenoidea and a new subfamily arrangement of a monophyletic Limacodidae: Crothaeminae (stat. rev.), Dalcerinae (stat. nov.), Chrysopolominae, and Limacodinae (stat. nov.). Two tribes recognized in Limacodinae, and previous subfamilies in Dalceridae and Chrysopolomidae are reduced to tribal rank. Synapomorphies of all life-stage characters are presented for Limacodidae and other family groups of Zygaenoidea. The total evidence topology is congruent with many features of egg, larval, and adult stage evolution. With new larval ontogenetic information, we present a hypothesis on how ontogeny and other modifications played a significant role in Limacodidae diversification through various defensive strategies.