Late Larval Stages Research Articles

Whole-Mount RNA In Situ Hybridization of Zebrafish Embryos.

A commonly employed technique in molecular biology to evaluate the temporal and spatial expression of a certain gene is in situ hybridization. This method is an effective strategy to construct synexpression groups, co-expressed genes acting in shared biological processes, and to find new members of genes engaged in the same signaling pathways to discover similar spatial and temporal expression patterns in zebrafish embryos. The major disadvantage of this method is that RNA probes can penetrate within 2days of post-fertilization embryos, and therefore, in later developmental stages, the probe can only reach the surface tissues. Further application of the method in histological sections will be required for a complete and accurate gene expression investigation. However, this method is highly effective at late embryogenesis and early larval stages for observing gene expression in endodermal derivatives and sensory organs. RNA probes for in situ hybridization can be prepared through in vitro transcription from plasmids carrying specific promoter elements and mRNA-specific cDNA, or an alternative polymerase chain reaction (PCR) method can be used through PCR amplification. This chapter describes the procedures for detecting gene expression in zebrafish embryos using whole-mount RNA in situ hybridization.

Young duplicate genes show developmental stage- and cell type-specific expression and function in Caenorhabditis elegans

Gene duplication produces the material that fuels evolutionary innovation. The "out-of-testis" hypothesis suggests that sperm competition creates selective pressure encouraging the emergence of new genes in male germline, but the somatic expression and function of the newly evolved genes are not well understood. We systematically mapped the expression of young duplicate genes throughout development in Caenorhabditis elegans using both whole-organism and single-cell transcriptomic data. Based on the expression dynamics across developmental stages, young duplicate genes fall into three clusters that are preferentially expressed in early embryos, mid-stage embryos, and late-stage larvae. Early embryonic genes are involved in protein degradation and develop essentiality comparable to the genomic average. In mid-to-late embryos and L4-stage larvae, young genes are enriched in intestine, epidermal cells, coelomocytes, and amphid chemosensory neurons. Their molecular functions and inducible expression indicate potential roles in innate immune response and chemosensory perceptions, which may contribute to adaptation outside of the sperm.

Development and application of a bioenergetics growth model for multiple early life stages of an ecologically important marine fish

Spatial and temporal variability in temperature and food availability are key drivers of growth of marine fishes. Growth during the early life stages (ELS's) is tightly coupled to survival, and in turn, can set year-class strength (i.e. annual recruitment) and overall stock productivity of populations and fished stocks. Ontogenetic changes in physiology, dietary preferences, and growth across ELS's can be accounted for within bioenergetics models, but existing models lack resolution within larval and early juvenile stages. We leveraged daily output from a coupled physical-biogeochemical model to force a highly resolved ontogenetic bioenergetics model parametrized for an ecologically important rockfish in the California Current System. Size-at-age predictions closely track empirical growth trajectories of the ELS's. Scenario testing revealed that growth performance is disproportionately driven by changes in temperature compared to food availability. We then expanded the model to incorporate spatial climatological differences in temperature and prey concentration and found that preflexion growth potential is maximized in areas of historical spawning, suggesting the timing and location of reproduction is an adaptive strategy that places larvae in habitat favorable for survival. Growth potential for late-stage larvae (postflexion) is greatest over a broad areal extent, implying that if a particle tracking algorithm was coupled to the bioenergetics model, a wide range of larval dispersal pathways would place postflexion larvae in habitat suitable for rapid growth. Finally, growth potential of pelagic juveniles is maximized over the continental shelf and shelf-break, aligning with high juvenile catch rates from a fisheries-independent survey. In summary, this study (i) serves as a proof of concept that a bioenergetics model with high ontogenetic resolution can reproduce life stage-specific growth trajectories even though the underlying physiology data for model parameterization is imperfect and (ii) can aid future studies aimed at understanding how ecosystem processes interact with ontogenetic growth and changes in year class strength of early life stages of marine fishes.

Bradysia (Sciara) coprophila larvae up-regulate DNA repair pathways and down-regulate developmental regulators in response to ionizing radiation.

The level of resistance to radiation and the developmental and molecular responses can vary between species, and even between developmental stages of one species. For flies (order: Diptera), prior studies concluded that the fungus gnat Bradysia (Sciara) coprophila (sub-order: Nematocera) is more resistant to irradiation-induced mutations that cause visible phenotypes than the fruit fly Drosophila melanogaster (sub-order: Brachycera). Therefore, we characterized the effects of and level of resistance to ionizing radiation on B. coprophila throughout its life cycle. Our data show that B. coprophila embryos are highly sensitive to even low doses of gamma-irradiation, whereas late-stage larvae can tolerate up to 80 Gy (compared to 40 Gy for D. melanogaster) and still retain their ability to develop to adulthood, though with a developmental delay. To survey the genes involved in the early transcriptional response to irradiation of B. coprophila larvae, we compared larval RNA-seq profiles with and without radiation treatment. The up-regulated genes were enriched for DNA damage response genes, including those involved in DNA repair, cell cycle arrest, and apoptosis, whereas the down-regulated genes were enriched for developmental regulators, consistent with the developmental delay of irradiated larvae. Interestingly, members of the PARP and AGO families were highly up-regulated in the B. coprophila radiation response. We compared the transcriptome responses in B. coprophila to the transcriptome responses in D. melanogaster from 3 previous studies: whereas pathway responses are highly conserved, specific gene responses are less so. Our study lays the groundwork for future work on the radiation responses in Diptera.

Could extremophilia be a life history strategy in late-stage larvae of Sparodon durbanensis (Family Sparidae)?

Microbialite pools are dynamic, heterogenous environments with extreme physico-chemical variability in terms of temporal temperature, oxygen and salinity fluctuations. Despite this habitat being hypothesised to be an ecological trap for larvae of marine estuary-dependent fishes, late-stage larvae and early juvenile White musselcracker (Sparodon durbanensis) ubiquitously occur in these pools. This study aimed to investigate the relationship between S. durbanensis density, developmental stage, total length and the physico-chemical environment using a multiple linear regression model analysis. Moreover, it aimed to further explore the potential nursery role of these extreme habitats. All larval and early juvenile stage S. durbanensis were collected with hand nets from pools at three representative microbialite sites on the warm temperate coast of South Africa from late summer to late autumn in 2021. A total of 63 pools were sampled with a total of 57 postflexion larvae and 162 early juvenile S. durbanensis specimens collected. Pool temperatures ranged from 16.6 to 37.3 °C with a mean of 24.5 °C, dissolved oxygen concentration ranged from 5.5 to 26.0 mg/L with a mean of 13.5 mg/L, and salinity ranged from 1.3 to 34.8 with a mean of 25.4. Temperature had a significant positive relationship with total length for early juvenile stages, while none of the other predictor variables showed evidence of a significant relationship with total length. Fish density showed a significant relationship with pool volume for both developmental stages where higher densities occurred in smaller pools that were also characterised by the most extreme physico-chemical variability. Late-stage larvae and early juveniles of this marine fish, that does not use estuaries as a nursery area, appears to complete the larval phase under extreme estuarine-like conditions in microbialite pools. Microbialite pools, as well as adjacent non-microbialite rocky shore pools appear to act as a micro-refuge, possibly improving growth rates and reducing the pelagic larval duration due to warmer water temperatures after which fish move into adjacent deeper, low shore pools and gullies. Due to the extreme conditions in microbialite pools, protection from predators during transition from larval to juvenile stages is also afforded.

First Report: Spodoptera Picta Guerin-Meneville, Armyworm on Swamp Lily (Crinum x powellii ‘Album’) in Bogor, Indonesia

Armyworms are very common and can attack all types of plants. Attacking ornamental plants such as flowers is no exception. This study aimed to determine the types of fall armyworms that attacked lilies in a Bogor, West Java house. It turned out to be an obvious symptom of the attack, the leaves became bald, attacked from the leaves to the base of the stem, and the dirt and the previous attack became wet. Armyworm larvae are dark grey, with eye-catching yellow stripes in the late larval stage. The larvae and adults were identified as a group of armyworms of Spodoptera picta (Lepidoptera: Noctuidae). They are common in Australia, and they attack lilies. Characteristics of the larvae were dark grey with a longitudinal black line, a striking yellow line that runs along the middle of the dorsal, and a Y shape in the head. The female of S. picta lays eggs in groups ranging from 50 to 218 eggs per egg group with an egg size of 1 mm. Lili armyworm larvae have 5 stages with larval sizes of 2, 5, 15, 28, and 46 mm respectively. Meanwhile, the size of the pupa and imago of S. picta was 23 and 20 mm, respectively. The management of S. picta armyworms needs to be done so as not to cause major damage and losses to ornamental flower crops.

First Report: Spodoptera Picta Guerin-Meneville, Armyworm on Swamp Lily (Crinum x powellii ‘Album’) in Bogor, Indonesia

Armyworms are very common and can attack all types of plants. Attacking ornamental plants such as flowers is no exception. This study aimed to determine the types of fall armyworms that attacked lilies in a Bogor, West Java house. It turned out to be an obvious symptom of the attack, the leaves became bald, attacked from the leaves to the base of the stem, and the dirt and the previous attack became wet. Armyworm larvae are dark grey, with eye-catching yellow stripes in the late larval stage. The larvae and adults were identified as a group of armyworms of Spodoptera picta (Lepidoptera: Noctuidae). They are common in Australia, and they attack lilies. Characteristics of the larvae were dark grey with a longitudinal black line, a striking yellow line that runs along the middle of the dorsal, and a Y shape in the head. The female of S. picta lays eggs in groups ranging from 50 to 218 eggs per egg group with an egg size of 1 mm. Lili armyworm larvae have 5 stages with larval sizes of 2, 5, 15, 28, and 46 mm respectively. Meanwhile, the size of the pupa and imago of S. picta was 23 and 20 mm, respectively. The management of S. picta armyworms needs to be done so as not to cause major damage and losses to ornamental flower crops.

Bursicon regulates wing expansion via PKA in the oriental fruit fly, Bactrocera dorsalis.

Bursicon is a heterodimeric neuropeptide that is involved in many physiological activities such as cuticle tanning, wing expansion, reproduction and immunity in insects. In this study, the role of bursicon in the wing expansion was investigated in Bactrocera dorsalis, an important invasive insect pest in agriculture. The cDNA sequences and deduced amino acids of bursicon genes (named BdBurs-α and BdBurs-β) were determined, and two proteins typically contained 11 cysteine residues in conserved positions that were highly conserved in other insect species. The spatiotemporal expressions of bursicon genes showed that higher expression occurred at the pupal, early adult stage and ovaries, and lower expression at the late larval stage and in wing tissue (8-day-old pupae). Dysfunction of bursicon genes by dsRNA microinjection into 5-day-old pupae reduced PKA (a downstream component of the bursicon pathway) activity and resulted in malformed adult wings. PKA inhibitor injection into 5-day-old pupae also resulted in similar phenotypes. Hematoxylin & eosin staining of the adult wing showed that RNAi and PKA inhibitor treatment reduced the thickness of the wing cuticle, which wing cuticle thickness were ≈50% thinner than in the control. Furthermore, the expression of hedgehog (Bdhh) (one of 10 tested genes related to wing development) was significantly upregulated after RNAi and PKA inhibitor application. The results indicate that bursicon plays a crucial role in the wing expansion of B. dorsalis, suggesting bursicon genes have potential to be the targets for B. dorsalis control. © 2023 Society of Chemical Industry.

Cydalima perspectalis (Walker) (Lepidoptera: Crambidae) üzerinde potansiyel biyolojik mücadele etmeni Bracon hebetor (Say) (Hymenoptera: Braconidae)’un etkinliği

Cydalima perspectalis (Walker, 1859) (Lepidoptera: Crambidae) is the main pest of boxwood fields in many countries. It has caused significant damage especially since 6-7 years in Türkiye. Chemical control is definitely not recommended against this pest because boxwoods are found in forest areas, parks and gardens. In this study, the potential use of the idibiont larval parasitoid Bracon hebetor (Say, 1836) (Hymenoptera: Braconidae) against the box tree moth was evaluated within the scope of biological control. Laboratory experiments were carried out with early and late instar larvae of C. perspectalis at 25±1°C, 60-70% R.H. and 16:8 (light: dark) conditions. The number of paralyzed and parasitized larvae, parasitism rate, number of eggs laid per female, rate of larvae and pupae were determined. Bracon hebetor, which has the ability to paralyze the host larvae, showed an insecticidal effect against the pest C. perspectalis. It was found that the number of paralyzed larvae (10.33±1.15) in early stage (3rd instar) larvae was higher than in late stage (5th instar) larvae (4.67±0.57). On the other hand, it was determined that the number of parasitized larvae (14.68±1.50) and eggs laid (7.50±0.32) was higher in late stage (5th instar) larvae. Parasitism rates were found to be 33% and 73% in early and late instar larvae, respectively. This detailed biological data has been obtained to for the first time. Bracon hebetor venom showed a high toxic effect on Cydalima larvae. The results of the study showed that the parasitoid may be recommended as an alternative to chemical control against the box tree moth.

Fatal Food: Silver-Coated Grain Particles Display Larvicidal Activity in Culex quinquefasciatus.

Mosquitoes pose a significant risk to millions of people worldwide since they can transmit pathogens. Current methods to control mosquito populations include the use of synthetic pesticides. Nanotechnology may be a solution to develop new mosquito control. However, one barrier to expanding the impact of nanomaterials is the ability to mass-produce the particles. Here, we report a novel hybrid particle synthesis combining micro- and nanoparticles using the coprecipitation technique with the potential for mass production. These particles may have applications as a mosquito larvacide. The particles reported here were designed using a microparticle zein polymer as the core and a nanoparticle silver as the active ingredient. The hybrid NPs reported here targeted a late-stage mosquito larvae and that resulted in a high larval mortality concentration (1.0 ppm, LC90) and suppression of pupal emergence at 0.1 ppm. This research demonstrates the efficacy of a plant-based core with a metal-based AI coating (AgNPs) against larval mosquitoes.

Minus-C odorant binding protein TcasOBP7G contributes to reproduction and defense against phytochemical in the red flour beetle, Tribolium castaneum

Insect odorant binding proteins (OBPs) paly an important role in the recognition and transmission signals. One odorant binding protein gene, TcasOBP7G, was significantly up-regulated after exposure to eucalyptol. Nevertheless, the causal role of TcasOBP7G in Tribolium castaneum is still unclear. Here, TcasOBP7G was cloned and characterized from T. castaneum, aligning with the typical characteristic of Minus-C OBPs. The analysis of gene expression profile showed that TcasOBP7G is highly expressed at late larval and adult stage, with particularly high expression levels in ovary and testis of adults, suggesting that TcasOBP7G might be associated with reproduction of T. castaneum. Furthermore, RNAi of TcasOBP7G led to a decrease in spawning, while the development of gonads was normal, suggesting that TcasOBP7G may medicate the reproduction of T. castaneum. Additionally, the mRNA level of TcasOBP7G was significantly increased after exposure to eucalyptol, and knockdown of TcasOBP7G resulted in an increased susceptibility of T. castaneum to eucalyptol, indicating that TcasOBP7G is associated with the tolerance of T. castaneum to eucalyptol. Altogether, these results indicate that TcasOBP7G is required for both reproduction and defense against phytochemical in T. castaneum. This study will provide a theoretical basis for the development of novel control strategies based on the olfaction of insects.

SeBLOS2 knockout via CRISPR/Cas9 leads to the loss of larval integument coloration in Spodoptera exigua (Lepidoptera: Noctuidae).

CRISPR/Cas9 technology is a precise and powerful tool for functionally exploring insect genes. The present study tested CRISPR/Cas9 as a way of undertaking effective gene mutagenesis in an important agricultural pest, the beet armyworm Spodoptera exigua. Based on a S. exigua transcriptome database, the entire complementary DNA sequence of SeBLOS2 encoding 140 amino acid residues was cloned. The gene was highly expressed in late larval stages (L3-L5). Using the CRISPR/Cas9 method, SeBLOS2 was knocked out by altering two sites in the coding region. This resulted in 70%-74% of the G0 generation (L4-L5) larvae displaying mosaic translucent integument. Four different mutations occurred at SeBLOS2-specific target sites, as demonstrated by further polymerase chain reaction-based genotypic analysis. Homozygote mutant L3 larvae were obtained in the G1 generation, with complete loss of white stripes and spots on their larval integument. These results demonstrate a crucial role of SeBLOS2 in integument pigmentation and suggest that the gene can act as a suitable nonlethal marker for functional research on genes in S. exigua and other Lepidopteran pests.

Immune markers in goats selected for reduced gastrointestinal nematode egg count under artificial infection conditions

This study aimed to further investigate the mechanisms responsible for inhibition of gastrointestinal nematode egg production in a line of Scottish Cashmere Goat selected for low faecal egg count. Animals were chosen as the lowest egg producers from a line selected for low egg output (selected group) with a second group from the wider herd based on high faecal egg count as controls. All animals were artificially infected with Teladorsagia circumcincta and Trichostrongylus vitrinus, then treated with anthelmintic and challenge infected prior to euthanasia and post mortem sampling.There was no effect on body weight between groups at any point in the study. Mean faecal egg count was reduced by 68 % in the selected group when compared with the controls. Circulating eosinophil counts were consistently elevated in the selected group, but this was only marginally significant (P = 0.045). Most of the circulating and tissue antibodies (IgG, IgA and IgE) measured were slightly elevated in the selected group but not significantly. Mucosal mast cells, eosinophils and globule leukocyte levels were higher in the abomasal and intestinal tissues in selected animals. Following challenge infection there was no difference in numbers of parasites, however there were more early stage parasite larvae and fewer late stage larvae in the both the abomasum and duodenum of the selected group compared with the unselected group, indicating some inhibition of parasite development.Overall, the study further demonstrated that selection based on low egg count has resulted in a line of goats producing significantly fewer parasites under identical infection with no effect on bodyweight. This appears to be associated with elevation in antibody and effector cells. Some evidence of host inhibition of parasite development was observed in the selected animals.

Growth patterns and feeding characteristics in early developmental stages of Takifugu rubripes cultured in a recirculating aquaculture system

The growth patterns and feeding characteristics of Takifugu rubripes at different developmental stages (0–50 days after hatching, DAH) were investigated under artificial breeding conditions using ecological and traditional biological methods. The results revealed rapid growth in head length, head height, and eye diameter from hatching to 50 DAH, and the inflection point for body length growth occurred at 27 DAH. From 27 to 50 DAH, the growth rate slowed. In the swimming organs, the inflection points for tail, pectoral, dorsal, and anal fin growth occurred from 29 to 31 DAH. The growth rate for fins slowed after this period, suggesting the preferential development of important organs related to sensation, locomotion, and feeding during early development, with varied developmental periods. An investigation of the feeding rhythm of T. rubripes larvae, juveniles, and young fish showed that feeding activities mainly occurred during the daytime, a behavior typical of daytime feeders. The average daily feeding rate of T. rubripes was 12.61% for the early-stage larvae at 6 DAH, 43.89% for the late-stage larvae at 15 DAH, 40.05% for juveniles at 30 DAH, and 28.46% for young fish at 46 DAH. In addition, the satiation time for juveniles tended to decrease with increasing age, whereas the time for digestion tended to increase. These data can be used as a reference for the daily feeding of T. rubripes at different developmental stages. Comprehensive understanding of the growth and feeding characteristics of early-stage T. rubripes developed in this study will be useful for the management of artificially bred fry and will aid in promoting the growth and survival rates of aquaculture fish larvae.

Fruitless is sex-differentially spliced and is important for the courtship behavior and development of silkmoth Bombyx mori

Sexual dimorphisms of the brain play essential roles in successful reproduction. Silkmoth Bombyx mori exhibits extensive sexual differences in sexual behavior, as well as their morphology. Although the neural circuits that transmit information about sex pheromone in the male brain are extensively analyzed, the molecular mechanisms that regulate their development are still elusive. In the present study, we focused on the silkmoth ortholog of fruitless (fru) as a candidate gene that regulates sexual dimorphisms of the brain. fru transcripts were expressed from multiple promoters in various tissues, and brain-specific transcripts were sex-specifically spliced, in a manner similar to Drosophila. Interestingly, fru was highly expressed in the adult female brain and the male larval testis. Analysis of CRISPR/Cas9-mediated fru knockout strains revealed that fru plays important roles in survival during late larval and pupal stages, testis development, and adult sexual behavior. fru mutant males exhibited highly reduced levels of courtship and low copulation rate, indicating that fru plays significant roles in the sexual behavior of silkmoths, although it is not absolutely necessary for copulation. In the fru mutant males, sexually dimorphic pattern of the odorant receptor expression was impaired, possibly causing the defects in courtship behavior. These results provide important clues to elucidate the development of sexual dimorphisms of silkmoth brains, as well as the evolution of fruitless gene in insects.

The C. elegans gene gvd-1 promotes late larval development and germ cell proliferation.

Limiting maternal resources necessitate deferring the development of adult-specific structures, notably the reproductive structures, to the postembryonic phase. These structures form postembryonically from blast cells generated during embryogenesis. A close coordination of developmental timing and pattern among the various postembryonic cell lineages is essential to form a functional adult. Here, we show that the C. elegans gene gvd-1 is essential for the development of several structures that form during the late larval stages. In gvd-1 mutant animals, blast cells that normally divide during the late larval stages (L3 and L4) fail to divide. In addition, germ cell proliferation is also severely reduced in these animals. Expression patterns of relevant reporter transgenes revealed a delay in G1/S transition in the vulval precursor cell P6.p and cytokinesis failure in seam cells in gvd-1 larvae. Our analyses of GVD-1::GFP transgenes indicate that GVD-1 is expressed in both soma and germ line, and functions in both. Sequence comparisons revealed that the sequence of gvd-1 is conserved only among nematodes, which does not support a broadly conserved housekeeping function for gvd-1. Instead, our results indicate a crucial role for gvd-1 that is specific to the larval development of nematodes.

Functional Analysis of a CTL-X-Type Lectin CTL16 in Development and Innate Immunity of Tribolium castaneum.

C-type lectins (CTLs) are a class of proteins containing carbohydrate recognition domains (CRDs), which are characteristic modules that recognize various glycoconjugates and function primarily in immunity. CTLs have been reported to affect growth and development and positively regulate innate immunity in Tribolium castaneum. However, the regulatory mechanisms of TcCTL16 proteins are still unclear. Here, spatiotemporal analyses displayed that TcCTL16 was highly expressed in late pupae and early adults. TcCTL16 RNA interference in early larvae shortened their body length and narrowed their body width, leading to the death of 98% of the larvae in the pupal stage. Further analysis found that the expression level of muscle-regulation-related genes, including cut, vestigial, erect wing, apterous, and spalt major, and muscle-composition-related genes, including Myosin heavy chain and Myosin light chain, were obviously down-regulated after TcCTL16 silencing in T. castaneum. In addition, the transcription of TcCTL16 was mainly distributed in the hemolymph. TcCTL16 was significantly upregulated after challenges with lipopolysaccharides, peptidoglycans, Escherichia coli, and Staphylococcus aureus. Recombinant CRDs of TcCTL16 bind directly to the tested bacteria (except Bacillus subtilis); they also induce extensive bacterial agglutination in the presence of Ca2+. On the contrary, after TcCTL16 silencing in the late larval stage, T. castaneum were able to develop normally. Moreover, the transcript levels of seven antimicrobial peptide genes (attacin2, defensins1, defensins2, coleoptericin1, coleoptericin2, cecropins2, and cecropins3) and one transcription factor gene (relish) were significantly increased under E. coli challenge and led to an increased survival rate of T. castaneum when infected with S. aureus or E. coli, suggesting that TcCTL16 deficiency could be compensated for by increasing AMP expression via the IMD pathways in T. castaneum. In conclusion, this study found that TcCTL16 could be involved in developmental regulation in early larvae and compensate for the loss of CTL function by regulating the expression of AMPs in late larvae, thus laying a solid foundation for further studies on T. castaneum CTLs.

Characterization of Glutamate-Gated Chloride Channel in Tribolium castaneum.

The glutamate-gated chloride channels (GluCls) play essential roles in signal transduction by regulating fast inhibitory synaptic transmission in the nervous system of invertebrates. While there is only one GluCl subunit in the insect, the diversity of insect GluCls is broadened by alternative splicing. In the present study, three TcGluCl variant genes were cloned from the red flour beetle Tribolium castaneum. Analysis of the characteristics of TcGluCls including sequence features, genomic structures, and alternative splicing revealed that TcGluCls had the typical structural features of GluCls and showed high homologies with the GluCls from other insect orders. The TcGluCl-encoding gene consists of nine exons and three variants (TcGluCl-3a, TcGluCl-3b, and TcGluCl-3c) were generated by the alternative splicing of exon 3, which was a highly conserved alternative splicing site in insect GluCls. Homology modeling of TcGluCl-3a showed that the exon 3 coding protein located at the N-terminal extracellular domain, and there were no steric clashes encountered between the exon 3 coding region and ivermectin/glutamate binding pocket, which indicated that the alternative splicing of exon 3 might have no impact on the binding of GluCls to glutamate and insecticide. In addition to the head tissue, TcGluCl-3a and TcGluCl-3c also had high expressions in the ovary and testis of T. castaneum, whereas TcGluCl-3b showed high expression in the midgut, suggesting the diverse physiological functions of TcGluCl variants in T. castaneum. The total TcGluCl and three variants showed the highest expression levels in the early stage larvae. The expressions of TcGluCl, TcGluCl-3b, and TcGluCl-3c were significantly increased from the late-stage larvae to the early stage pupae and indicated that the TcGluCl might be involved in the growth and development of T. castaneum. These results are helpful to further understand the molecular characteristics of insect GluCls and provide foundations for studying the specific function of the GluCl variant.

Investigating the unaccounted ones: insights on age-dependent reproductive loss in a viviparous fly.

Most empirical and theoretical studies on reproductive senescence focus on observable attributes of offspring produced, such as size or postnatal survival. While harder to study, an important outcome of reproduction for a breeding individual is whether a viable offspring is produced at all. While prenatal mortality can sometimes be directly observed, this can also be indicated through an increase in the interval between offspring production. Both direct reproductive loss and presumed losses have been found to increase in older females across several species. Here, we study such reproductive loss (or "abortion") in tsetse, a viviparous and relatively long-lived fly with high maternal allocation. We consider how age-dependent patterns of abortion depend on the developmental stage of offspring and find that, as per previous laboratory studies, older females have higher rates of abortion at the late-larval stage, while egg-stage abortions are high both for very young and older females. We track the reproductive output of individual females and find little evidence that experiencing an abortion is an adaptive strategy to improve future reproductive outcomes. After an abortion, females do not generally take less time to produce their next offspring, these offspring are not larger, and there is no sex bias towards females, the sex with presumed higher fitness returns (being slightly larger and longer-lived than males, and with high insemination rates). Abortion rates are higher for breeding females experiencing stress, measured as nutritional deprivation, which echoes previous work in tsetse and other viviparous species, i.e., humans and baboons. We discuss our results in the context of studies on reproductive loss across taxa and argue that this is an important yet often overlooked reproductive trait which can vary with maternal age and can also depend on environmental stressors.

A rapidly evolving single copy histone H1 variant is associated with male fertility in a parasitoid wasp.

The linker histone H1 binds to the nucleosome core particle at the site where DNA enters and exits, and facilitates folding of the nucleosomes into a higher-order chromatin structure in eukaryotes. Additionally, some variant H1s promote specialized chromatin functions in cellular processes. Germline-specific H1 variants have been reported in some model species with diverse roles in chromatin structure changes during gametogenesis. In insects, the current understanding of germline-specific H1 variants comes mainly from the studies in Drosophila melanogaster, and the information on this set of genes in other non-model insects remains largely unknown. Here, we identify two H1 variants (PpH1V1 and PpH1V2) that are specifically predominantly expressed in the testis of the parasitoid wasp Pteromalus puparum. Evolutionary analyses suggest that these H1 variant genes evolve rapidly, and are generally maintained as a single copy in Hymenoptera. Disruption of PpH1V1 function in the late larval stage male by RNA interference experiments has no phenotype on spermatogenesis in the pupal testis, but results in abnormal chromatin structure and low sperm fertility in the adult seminal vesicle. In addition, PpH1V2 knockdown has no detectable effect on spermatogenesis or male fertility. Collectively, our discovery indicates distinct functions of male germline-enriched H1 variants between parasitoid wasp Pteromalus and Drosophila, providing new insights into the role of insect H1 variants in gametogenesis. This study also highlights the functional complexity of germline-specific H1s in animals.