RESUMO
Telomerase and telomerase-generated telomeric DNA sequences are widespread throughout eukaryotes, yet they are not universal. Neither telomerase nor the simple DNA repeats associated with telomerase have been found in some plant and animal species. Telomerase was likely lost from Diptera before the divergence of Diptera and Siphonaptera, some 260 million years ago. Even so, Diptera is one of the most successful animal orders, making up 11% of known animal species. In addition, many species of Coleoptera and Hemiptera seem to lack canonical telomeric repeats at their chromosome ends. These and other insects that appear to lack canonical terminal repeat sequences account for another 10-15% of animal species. Conversely, the silk moth Bombyx mori maintains canonical telomeric sequences at its chromosome ends but seems to lack a functional telomerase. We speculate that a telomere-specific capping complex that recognizes the telomeric repeats and protects chromosome ends is the determining factor in maintaining canonical telomeric sequences and that telomerase is an early and efficacious mechanism for satisfying the needs of capping complex. There are alternate mechanisms for maintaining chromosome ends that do not depend on telomerase, such as recombination found in some human cancer cells and yeast mutants. These mechanisms may maintain the canonical telomeric repeats or allow the terminal sequence to evolve when specificity of the capping complex for terminal repeat sequences is weak.
Assuntos
Evolução Molecular , Deleção de Genes , Insetos/enzimologia , Telomerase/genética , Telômero/metabolismo , Animais , Recombinação Homóloga , Insetos/genética , Sequências Repetidas TerminaisRESUMO
PURPOSE OF REVIEW: This review addresses the most recent developments on cockroach allergen research in relation to allergic diseases, especially asthma. RECENT FINDINGS: The number of allergens relevant to cockroach allergy has recently expanded considerably up to 12 groups. New X-ray crystal structures of allergens from groups 1, 2, and 5 revealed interesting features with implications for allergen standardization, sensitization, diagnosis, and therapy. Cockroach allergy is strongly associated with asthma particularly among children and young adults living in inner-city environments, posing challenges for disease control. Environmental interventions targeted at reducing cockroach allergen exposure have provided conflicting results. Immunotherapy may be a way to modify the natural history of cockroach allergy and decrease symptoms and asthma severity among sensitized and exposed individuals. The new information on cockroach allergens is important for the assessment of allergen markers of exposure and disease, and for the design of immunotherapy trials.
Assuntos
Alérgenos/análise , Asma/etiologia , Baratas/imunologia , Exposição Ambiental/análise , Animais , Criança , HumanosRESUMO
Since the discovery that Der p 1 is a cysteine protease, the role of proteolytic activity in allergic sensitization has been explored. There are many allergens with proteolytic activity; however, exposure from dust mites is not limited to allergens. In this paper, genomic, transcriptomic and proteomic data on Dermatophagoides pteronyssinus (DP) was mined for information regarding the complete degradome of this house dust mite. D. pteronyssinus has more proteases than the closely related Acari, Dermatophagoides farinae (DF) and Sarcoptes scabiei (SS). The group of proteases in D. pteronyssinus is found to be more highly transcribed than the norm for this species. The distribution of protease types is dominated by the cysteine proteases like Der p 1 that account for about half of protease transcription by abundance, and Der p 1 in particular accounts for 22% of the total protease transcripts. In an analysis of protease stability, the group of allergens (Der p 1, Der p 3, Der p 6, and Der p 9) is found to be more stable than the mean. It is also statistically demonstrated that the protease allergens are simultaneously more highly expressed and more stable than the group of D. pteronyssinus proteases being examined, consistent with common assumptions about allergens in general. There are several significant non-allergen outliers from the normal group of proteases with high expression and high stability that should be examined for IgE binding. This paper compiles the first holistic picture of the D. pteronyssinus degradome to which humans may be exposed.
Assuntos
Antígenos de Dermatophagoides/análise , Proteínas de Artrópodes/análise , Cisteína Endopeptidases/análise , Dermatophagoides pteronyssinus/química , Serina Endopeptidases/análise , Alérgenos/análise , Alérgenos/genética , Sequência de Aminoácidos , Animais , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes/genética , Cisteína Endopeptidases/genética , Dermatophagoides pteronyssinus/genética , Estabilidade Enzimática , Filogenia , Alinhamento de Sequência , Serina Endopeptidases/genéticaRESUMO
In most mammals, the Zfp36 gene family consists of three conserved members, with a fourth member, Zfp36l3, present only in rodents. The ZFP36 proteins regulate post-transcriptional gene expression at the level of mRNA stability in organisms from humans to yeasts, and appear to be expressed in all major groups of eukaryotes. In Mus musculus, Zfp36l3 expression is limited to the placenta and yolk sac, and is important for overall fecundity. We sequenced the Zfp36l3 gene from more than 20 representative species, from members of the Muridae, Cricetidae and Nesomyidae families. Zfp36l3 was not present in Dipodidae, or any families that branched earlier, indicating that this gene is exclusive to the Muroidea superfamily. We provide evidence that Zfp36l3 arose by retrotransposition of an mRNA encoded by a related gene, Zfp36l2 into an ancestral rodent X chromosome. Zfp36l3 has evolved rapidly since its origin, and numerous modifications have developed, including variations in start codon utilization, de novo intron formation by mechanisms including a nested retrotransposition, and the insertion of distinct repetitive regions. One of these repeat regions, a long alanine rich-sequence, is responsible for the full-time cytoplasmic localization of Mus musculus ZFP36L3. In contrast, this repeat sequence is lacking in Peromyscus maniculatus ZFP36L3, and this protein contains a novel nuclear export sequence that controls shuttling between the nucleus and cytosol. Zfp36l3 is an example of a recently acquired, rapidly evolving gene, and its various orthologues illustrate several different mechanisms by which new genes emerge and evolve.
Assuntos
Evolução Molecular , Roedores/genética , Tristetraprolina/genética , Animais , Núcleo Celular/genética , Feminino , Humanos , Íntrons , Muridae/genética , Peromyscus/genética , Filogenia , Placenta/metabolismo , Gravidez , Proteínas/genética , RNA Mensageiro , Sequências Repetitivas de Ácido Nucleico , RetroelementosAssuntos
Alérgenos , Hipersensibilidade , Poeira , Humanos , Hipersensibilidade/epidemiologia , Hipersensibilidade/etiologia , PólenAssuntos
Alérgenos/genética , Antígenos de Dermatophagoides/genética , Dermatophagoides farinae/genética , Dermatophagoides pteronyssinus/genética , Genoma , Sequência de Aminoácidos , Animais , Dermatophagoides farinae/imunologia , Dermatophagoides pteronyssinus/imunologia , Marcadores Genéticos , Isoformas de Proteínas , Homologia de Sequência de Aminoácidos , Sequenciamento Completo do GenomaRESUMO
Myocoptes musculinus is a common ectoparasite of wild mice and is occasionally found on research mice. Infestations of research mice are often subclinical but can cause severe dermatitis. Perhaps more importantly, infestations can cause immunologic reactions that may alter research outcomes, and most animal research facilities strive to prevent or eliminate mites from their mouse colonies. M. musculinus infestations are currently detected by using microscopic evaluation of the fur and skin and PCR assays of pelt swabs targeting the rRNA genes of this mite. In our facility, we encountered multiple, false-positive 18S rRNA PCR results from a closed mouse colony. We could not identify the source of the false positives even after performing PCR analysis of other Myocoptes gene targets using assays developed from the few other target genomic sequences available for M. musculinus or Myocoptes japonensis in public databases. This situation highlighted the limited genetic resources available for development of diagnostic tests specific for this ectoparasite. To expand the available genetic resources, we generated a metagenome of M. musculinus derived by sequencing from fur plucks of an infected mouse. We also determined the completeness of this metagenome and compared it with those of related mites.
Assuntos
Infestações por Ácaros , Ácaros , Animais , Camundongos , Infestações por Ácaros/diagnóstico , Infestações por Ácaros/veterinária , Pele , Reação em Cadeia da PolimeraseRESUMO
Escherichia coli pathogenic variants (pathovars) are generally characterized by defined virulence traits and are susceptible to the evolution of hybridized identities due to the considerable plasticity of the E. coli genome. We have isolated a strain from a purified diet intended for research animals that further demonstrates the ability of E. coli to acquire novel genetic elements leading potentially to emergent new pathovars. Utilizing next generation sequencing to obtain a whole genome profile, we report an atypical strain of E. coli, EcoFA807-17, possessing a tetrathionate reductase (ttr) operon, which enables the utilization of tetrathionate as an electron acceptor, thus facilitating respiration in anaerobic environments such as the mammalian gut. The ttr operon is a potent virulence factor for several enteric pathogens, most prominently Salmonella enterica. However, the presence of chromosomally integrated tetrathionate reductase genes does not appear to have been previously reported in wild-type E. coli or Shigella. Accordingly, it is possible that the appearance of this virulence factor may signal the evolution of new mechanisms of pathogenicity in E. coli and Shigella and may potentially alter the effectiveness of existing assays using tetrathionate reductase as a unique marker for the detection of Salmonella enterica.
Assuntos
Escherichia coli , Shigella , Escherichia coli/genética , Fatores de Virulência/genéticaRESUMO
Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9 + million SARS-CoV-2 sequences revealed mutations across Nsp15's three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants in vivo.
Assuntos
COVID-19 , Endorribonucleases , SARS-CoV-2 , Endorribonucleases Específicas de Uridilato , Proteínas não Estruturais Virais , COVID-19/virologia , Endorribonucleases/química , Endorribonucleases/genética , Humanos , Proteínas Recombinantes/química , SARS-CoV-2/enzimologia , Endorribonucleases Específicas de Uridilato/química , Endorribonucleases Específicas de Uridilato/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9+ million SARS-CoV-2 sequences revealed mutations across Nsp15â™s three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants in vivo .
RESUMO
Telomeres in Drosophila melanogaster, which have inspired a large part of Sergio Pimpinelli work, are similar to those of other eukaryotes in terms of their function. Yet, their length maintenance relies on the transposition of the specialized retrotransposons Het-A, TART, and TAHRE, rather than on the activity of the enzyme telomerase as it occurs in most other eukaryotic organisms. The length of the telomeres in Drosophila thus depends on the number of copies of these transposable elements. Our previous work has led to the isolation of a dominant mutation, Tel1, that caused a several-fold elongation of telomeres. In this study, we molecularly identified the Tel1 mutation by a combination of transposon-induced, site-specific recombination and next-generation sequencing. Recombination located Tel1 to a 15 kb region in 92A. Comparison of the DNA sequence in this region with the Drosophila Genetic Reference Panel of wild-type genomic sequences delimited Tel1 to a 3 bp deletion inside intron 8 of Ino80. Furthermore, CRISPR/Cas9-induced deletions surrounding the same region exhibited the Tel1 telomere phenotype, confirming a strict requirement of this intron 8 gene sequence for a proper regulation of Drosophila telomere length.
Assuntos
Drosophila melanogaster , Drosophila , Animais , Drosophila/genética , Drosophila melanogaster/genética , Produtos do Gene gag/genética , Telômero/genética , Mutação/genéticaRESUMO
In many cases, genes for commonly used genetic markers in model organisms have not been identified; therefore, it is of interest to identify the causative genes. Whole-genome sequencing was used to identify potential causative mutations for a col-4 allele of Neurospora crassa.
RESUMO
The inositol pyrophosphates (PP-InsPs) are a unique subgroup of intracellular signals with diverse functions, many of which can be viewed as reflecting an overarching role in metabolic homeostasis. Thus, considerable attention is paid to the enzymes that synthesize and metabolize the PP-InsPs. One of these enzyme families - the diphosphoinositol pentakisphosphate kinases (PPIP5Ks) - provides an extremely rare example of separate kinase and phosphatase activities being present within the same protein. Herein, we review the current state of structure/function insight into the PPIP5Ks, the separate specialized activities of the two metazoan PPIP5K genes, and we describe a phylogenetic analysis that places PPIP5K evolutionary origin within the Excavata, the very earliest of eukaryotes. These different aspects of PPIP5K biology are placed in the context of a single, overriding question. Why are they bifunctional: i.e., what is the particular significance of the ability to turn PP-InsP signaling on or off from two separate 'switches' in a single protein?
Assuntos
Evolução Molecular , Fosfatos de Inositol , Fosfotransferases (Aceptor do Grupo Fosfato) , Transdução de Sinais , Animais , Humanos , Fosfatos de Inositol/genética , Fosfatos de Inositol/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismoRESUMO
Genomes of tens of thousands of SARS-CoV2 isolates have been sequenced across the world and the total number of changes (predominantly single base substitutions) in these isolates exceeds ten thousand. We compared the mutational spectrum in the new SARS-CoV-2 mutation dataset with the previously published mutation spectrum in hypermutated genomes of rubella-another positive single stranded (ss) RNA virus. Each of the rubella virus isolates arose by accumulation of hundreds of mutations during propagation in a single subject, while SARS-CoV-2 mutation spectrum represents a collection events in multiple virus isolates from individuals across the world. We found a clear similarity between the spectra of single base substitutions in rubella and in SARS-CoV-2, with C to U as well as A to G and U to C being the most prominent in plus strand genomic RNA of each virus. Of those, U to C changes universally showed preference for loops versus stems in predicted RNA secondary structure. Similarly, to what was previously reported for rubella virus, C to U changes showed enrichment in the uCn motif, which suggested a subclass of APOBEC cytidine deaminase being a source of these substitutions. We also found enrichment of several other trinucleotide-centered mutation motifs only in SARS-CoV-2-likely indicative of a mutation process characteristic to this virus. Altogether, the results of this analysis suggest that the mutation mechanisms that lead to hypermutation of the rubella vaccine virus in a rare pathological condition may also operate in the background of the SARS-CoV-2 viruses currently propagating in the human population.
Assuntos
Betacoronavirus/genética , Genoma Viral , RNA Viral/genética , Vírus da Rubéola/genética , COVID-19 , Infecções por Coronavirus/virologia , Citidina Desaminase/genética , Bases de Dados Genéticas , Evolução Molecular , Humanos , Mutação , Pandemias , Pneumonia Viral/virologia , SARS-CoV-2RESUMO
Genomes of tens of thousands of SARS-CoV2 isolates have been sequenced across the world and the total number of changes (predominantly single base substitutions) in these isolates exceeds ten thousand. We compared the mutational spectrum in the new SARS-CoV-2 mutation dataset with the previously published mutation spectrum in hypermutated genomes of rubella - another positive single stranded (ss) RNA virus. Each of the rubella isolates arose by accumulation of hundreds of mutations during propagation in a single subject, while SARS-CoV-2 mutation spectrum represents a collection events in multiple virus isolates from individuals across the world. We found a clear similarity between the spectra of single base substitutions in rubella and in SARS-CoV-2, with C to U as well as A to G and U to C being the most prominent in plus strand genomic RNA of each virus. Of those, U to C changes universally showed preference for loops versus stems in predicted RNA secondary structure. Similarly, to what was previously reported for rubella, C to U changes showed enrichment in the uCn motif, which suggested a subclass of APOBEC cytidine deaminase being a source of these substitutions. We also found enrichment of several other trinucleotide-centered mutation motifs only in SARS-CoV-2 - likely indicative of a mutation process characteristic to this virus. Altogether, the results of this analysis suggest that the mutation mechanisms that lead to hypermutation of the rubella vaccine virus in a rare pathological condition may also operate in the background of the SARS-CoV-2 viruses currently propagating in the human population.
RESUMO
Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in metabolism, DNA repair, and aging. However, how NAD metabolism is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer resistance to inhibitors of NAMPT, the rate-limiting enzyme in the amidated NAD salvage pathway, in cancer cells and xenograft tumors. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism.
Assuntos
Amidas/metabolismo , Vias Biossintéticas , Mamíferos/microbiologia , Mycoplasma/fisiologia , NAD/metabolismo , Administração Oral , Animais , Linhagem Celular Tumoral , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Metabolismo Energético , Feminino , Microbioma Gastrointestinal , Humanos , Masculino , Metaboloma , Camundongos Endogâmicos C57BL , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Nicotinamidase/metabolismo , Mononucleotídeo de Nicotinamida/administração & dosagem , Mononucleotídeo de Nicotinamida/química , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/metabolismo , Compostos de Piridínio/metabolismoRESUMO
Choline is an essential nutrient for humans, though some of the requirement can be met by endogenous synthesis catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). Premenopausal women are relatively resistant to choline deficiency compared with postmenopausal women and men. Studies in animals suggest that estrogen treatment can increase PEMT activity. In this study we investigated whether the PEMT gene is regulated by estrogen. PEMT transcription was increased in a dose-dependent manner when primary mouse and human hepatocytes were treated with 17-beta-estradiol for 24 h. This increased message was associated with an increase in protein expression and enzyme activity. In addition, we report a region that contains a perfect estrogen response element (ERE) approximately 7.5 kb from the transcription start site corresponding to transcript variants NM_007169 and NM-008819 of the human and murine PEMT genes, respectively, three imperfect EREs in evolutionarily conserved regions and multiple imperfect EREs in nonconserved regions in the putative promoter regions. We predict that both the mouse and human PEMT genes have three unique transcription start sites, which are indicative of either multiple promoters and/or alternative splicing. This study is the first to explore the underlying mechanism of why dietary requirements for choline vary with estrogen status in humans.
Assuntos
Estradiol/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/enzimologia , Fosfatidiletanolamina N-Metiltransferase/genética , Processamento Alternativo , Animais , Células Cultivadas , Deficiência de Colina/enzimologia , Feminino , Hepatócitos/citologia , Humanos , Masculino , Camundongos , Pós-Menopausa , Pré-Menopausa , Regiões Promotoras Genéticas , RNA Mensageiro/genéticaRESUMO
MicroRNAs (miRNAs) are small (18-24 nt) endogenous RNAs found across diverse phyla involved in posttranscriptional regulation, primarily downregulation of mRNAs. Experimentally determining miRNA-mRNA interactions can be expensive and time-consuming, making the accurate computational prediction of miRNA targets a high priority. Since miRNA-mRNA base pairing in mammals is not perfectly complementary and only a fraction of the identified motifs are real binding sites, accurately predicting miRNA targets remains challenging. The limitations and bottlenecks of existing algorithms and approaches are discussed in this chapter.A new miRNA-mRNA interaction algorithm was implemented in Python (TargetFind) to capture three different modes of association and to maximize detection sensitivity to around 95% for mouse (mm9) and human (hg19) reference data. For human (hg19) data, the prediction accuracy with any one feature among evolutionarily conserved score, multiple targets in a UTR or changes in free energy varied within a close range from 63.5% to 66%. When the results of these features are combined with majority voting, the expected prediction accuracy increases to 69.5%. When all three features are used together, the average best prediction accuracy with tenfold cross validation from the classifiers naïve Bayes, support vector machine, artificial neural network, and decision tree were, respectively, 66.5%, 67.1%, 69%, and 68.4%. The results reveal the advantages and limitations of these approaches.When comparing different sets of features on their strength in predicting true hg19 targets, evolutionarily conserved score slightly outperformed all other features based on thermostability, and target multiplicity. The sophisticated supervised learning algorithms did not improve the prediction accuracy significantly compared to a simple threshold based approach on conservation score or combining the results of each feature with majority agreements. The targets from randomly generated UTRs behaved similar to that of noninteracting pairs with respect to changes in free energy. Availability of additional experimental data describing noninteracting pairs will advance our understanding of the characteristics and the factors positively and negatively influencing these interactions.
Assuntos
Regulação da Expressão Gênica , Genômica/métodos , MicroRNAs/genética , RNA Mensageiro/genética , Algoritmos , Animais , Evolução Molecular , Humanos , Aprendizado de Máquina , MicroRNAs/química , Estabilidade de RNA , RNA Mensageiro/química , Software , Termodinâmica , Regiões não TraduzidasRESUMO
The major allergen domain (MA) is widely distributed in insects. The crystal structure of a single Bla g 1 MA revealed a novel protein fold in which the fundamental structure was a duplex of two subsequences (monomers), which had diverged over time. This suggested that the evolutionary origin of the MA structure may have been a homodimer of this smaller subsequence. Using publicly available genomic data, the distribution of the basic unit of this class of proteins was determined to better understand its evolutionary history. The duplication and divergence is examined at three distinct levels of resolution: 1) within the orders Diptera and Hymenoptera, 2) within one genus Drosophila, and 3) within one species Aedes aegypti. Within the family Culicidae, we have found two separate occurrences of monomers as independent genes. The organization of the gene family in A. aegypti shows a common evolutionary origin for its monomer and several closely related MAs. Molecular modeling of the A. aegypti monomer with the unique Bla g 1 fold confirms the distant evolutionary relationship and supports the feasibility of homodimer formation from a single monomer. RNAseq data for A. aegypti confirms that the monomer is expressed in the mosquito similar to other A. aegypti MAs after a blood meal. Together, these data support the contention that the detected monomer shares similar functional characteristics to related MAs in other insects. An extensive search for this domain outside of Insecta confirms that the MAs are restricted to insects.