Immunocytochemical studies on peptidergic neurons in the Colorado potato beetle and some other insect species

This thesis describes the distribution, numbers, and morphology of peptidergic neurons and neurosecretory cells in the Colorado potato beetle, as detected with immunocytochemistry with antisera to various regulatory peptides from vertebrates, as well as to the molluscan cardioexcitatory peptide FMRFamide, and to the insect neuropeptide proctolin.In chapter one, a description is given of peptidergic neurons in the Colorado potato beetle which are immunoreactive with antisera to FMRFamide and bovine pancreatic polypeptide. Results from cross-adsorption experiments suggest that the antisera to FMRFamide and bovine pancreatic polypeptide cross-react and reveal the same substance(s) in the Colorado potato beetle. Some of the immunoreactive neurons branch extensively in the neuropile, suggesting that the immunoreactive substance is used as a transmitter or modulator. The corpus cardiacum contains numerous immunoreactive axon terminals from neurosecretory cells located in the suboesophageal ganglion. These cells probably use the peptide as a hormone.In chapter two, ten different antisera were used to differentiate between neurons in the neuroendocrine system of the Colorado potato beetle. Four antisera to vasopressin, three to oxytocin and one to vasotocin, neurophysin-1 and neurophysin-2 each were used. All antisera revealed immunoreactive cells, except for the two neurophysin antisera. The antisera to vasopressin all gave different results. Whereas one antiserurn revealed only a single neuron pair, the other three antisera revealed additional groups of immunoreactive cells. The three oxytocin antisera also gave different results. It was further found that the fixation procedure also greatly influenced the immunocytochemical results. It was concluded that several vasopressin and oxytocin immunoreactive peptides are present in the Colorado potato beetle, all with a different degree of resemblance to vasopressin and oxytocin.In chapter three, it was investigated whether immunocytochemical techniques could be used for the demonstration of homologous neurosecretory cells in the suboesophageal ganglion of the Colorado potato beetle and of the migratory locust. The antisera were anti-FMRFamide, anti-bovine pancreatic polypeptide, two antisera to vasopressin, which all had been used before in the work described in chapters 1 and 2, and an antiserurn to α-MSH. It is shown that neurosecretory cells are present in the suboesophageal ganglion of both species and that they occur in similar locations and have the same immunoreactivity. This suggests that the two species contain homologous neurosecretory cells. It is speculated that they also have similar functions.In chapter four, the concept of homologous neurons is further developed and ten different insect species were examined for the presence of neurons immunoreactive with a specific antiserurn to gastrin-releasing peptide. Immunoreactive neurons were revealed in seven species, belonging to seven orders. The concentration of antiserurn required to demonstrate immunoreactive cells was much higher in insects than in chicken proventriculus, known to contain gastrin-releasing peptide. Some immunoreactive neurons were found in the same numbers and approximately the same locations within the nervous system of different insect species. This suggests that the substances in these neurons have been relatively stable during evolution and that these neurons are homologous.In chapter five, a large number of antisera to various regulatory vertebrate peptides was tested on the Colorado potato beetle, to reveal peptidergic neurons and neurosecretory cells and to differentiate between these cells. Immunoreactive cells were revealed by antisera to ACTH, gastrin, cholecystokinin, α-endorphin, β-endorphin, γl-MSH, insulin, human calcitonin, motilin, growth hormone, somatostatin, corticotropin-releasing factor, ovine prolactin and rat prolactin. Some of these immunoreactive cells seem to function as neurons, whereas others function as neurosecretory cells. It is postulated that if the part of the substance recognized by the antiserurn is of physiological importance to the insect, that part is retained in several species. The antiserurn should then reveal homologous neurons in different insect species. Given the fact that insect species differ widely in their immunocytochemical responses, the criterion of staining of probably homologous neurons offers some help in separating relevant from irrelevant immunoreactions. The immunocytochemical data are evaluated according to this concept.In chapter six, neurons in the central and peripheral nervous system of the Colorado potato beetle were described which contain substances immunoreactive with antiserurn to the insect neuropeptide proctolin. This peptide was originally isolated from cockroaches, in which it stimulates contractions of the gut musculature. Immunoreactive axon terminals were also found on the muscles of the fore- and hindgut, and abdominal segments, as well as in the vas deferens of the testis. Furthermore, the nervous system was extracted to investigate whether proctolin-like bioactivity could be demonstrated. A proctolin-like bioactive peptide was demonstrated, that behaved chromatographically like proctolin. This suggests that at least some of the immunocytochemically demonstrated proctolin is identical with or at least similar to proctolin

Clustered mutations in the GRIK2 kainate receptor subunit gene underlie diverse neurodevelopmental disorders

Kainate receptors (KARs) are glutamate-gated cation channels with diverse roles in the central nervous system. Bi-allelic loss of function of the KAR-encoding gene GRIK2 causes a nonsyndromic neurodevelopmental disorder (NDD) with intellectual disability and developmental delay as core features. The extent to which mono-allelic variants in GRIK2 also underlie NDDs is less understood because only a single individual has been reported previously. Here, we describe an additional eleven individuals with heterozygous de novo variants in GRIK2 causative for neurodevelopmental deficits that include intellectual disability. Five children harbored recurrent de novo variants (three encoding p.Thr660Lys and two p.Thr660Arg), and four children and one adult were homozygous for a previously reported variant (c.1969G>A [p.Ala657Thr]). Individuals with shared variants had some overlapping behavioral and neurological dysfunction, suggesting that the GRIK2 variants are likely pathogenic. Analogous mutations introduced into recombinant GluK2 KAR subunits at sites within the M3 transmembrane domain (encoding p.Ala657Thr, p.Thr660Lys, and p.Thr660Arg) and the M3-S2 linker domain (encoding p.Ile668Thr) had complex effects on functional properties and membrane localization of homomeric and heteromeric KARs. Both p.Thr660Lys and p.Thr660Arg mutant KARs exhibited markedly slowed gating kinetics, similar to p.Ala657Thr-containing receptors. Moreover, we observed emerging genotype-phenotype correlations, including the presence of severe epilepsy in individuals with the p.Thr660Lys variant and hypomyelination in individuals with either the p.Thr660Lys or p.Thr660Arg variant. Collectively, these results demonstrate that human GRIK2 variants predicted to alter channel function are causative for early childhood development disorders and further emphasize the importance of clarifying the role of KARs in early nervous system development.Genetics of disease, diagnosis and treatmen

Clustered mutations in the GRIK2 kainate receptor subunit gene underlie diverse neurodevelopmental disorders (vol 108, pg 1692, 2021)

Neurolog

Genome-enabled insights into the biology of thrips as crop pests

Background The western flower thrips,Frankliniella occidentalis(Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present theF. occidentalisdraft genome assembly and official gene set.Results We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which similar to 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta.Conclusions Analysis of theF. occidentalisgenome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species.Animal science

The Prospective Dutch Colorectal Cancer (PLCRC) cohort: real-world data facilitating research and clinical care

Real-world data (RWD) sources are important to advance clinical oncology research and evaluate treatments in daily practice. Since 2013, the Prospective Dutch Colorectal Cancer (PLCRC) cohort, linked to the Netherlands Cancer Registry, serves as an infrastructure for scientific research collecting additional patient-reported outcomes (PRO) and biospecimens. Here we report on cohort developments and investigate to what extent PLCRC reflects the “real-world”. Clinical and demographic characteristics of PLCRC participants were compared with the general Dutch CRC population (n = 74,692, Dutch-ref). To study representativeness, standardized differences between PLCRC and Dutch-ref were calculated, and logistic regression models were evaluated on their ability to distinguish cohort participants from the Dutch-ref (AU-ROC 0.5 = preferred, implying participation independent of patient characteristics). Stratified analyses by stage and time-period (2013–2016 and 2017–Aug 2019) were performed to study the evolution towards RWD. In August 2019, 5744 patients were enrolled. Enrollment increased steeply, from 129 participants (1 hospital) in 2013 to 2136 (50 of 75 Dutch hospitals) in 2018. Low AU-ROC (0.65, 95% CI: 0.64–0.65) indicates limited ability to distinguish cohort participants from the Dutch-ref. Characteristics that remained imbalanced in the period 2017–Aug’19 compared with the Dutch-ref were age (65.0 years in PLCRC, 69.3 in the Dutch-ref) and tumor stage (40% stage-III in PLCRC, 30% in the Dutch-ref). PLCRC approaches to represent the Dutch CRC population and will ultimately meet the current demand for high-quality RWD. Efforts are ongoing to improve multidisciplinary recruitment which will further enhance PLCRC’s representativeness and its contribution to a learning healthcare system

Partitioning the Heritability of Tourette Syndrome and Obsessive Compulsive Disorder Reveals Differences in Genetic Architecture

The direct estimation of heritability from genome-wide common variant data as implemented in the program Genome-wide Complex Trait Analysis (GCTA) has provided a means to quantify heritability attributable to all interrogated variants. We have quantified the variance in liability to disease explained