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Transcriptional regulation of the human PAX6 gene promoter.

PAX6, a member of the highly conserved paired-type homeobox gene family, is expressed in a spatially and temporally restricted pattern during early embryogenesis, and its mutation is responsible for human aniridia. Here we examined the transcriptional regulation of the PAX6 gene by transient transfection assays and identified multiple cis-regulatory elements that function differently in different cell lines. The transcriptional initiation site was identified by RNase protection and primer extension assay. Examination of the genomic DNA sequence indicated that the PAX6 promoter has a TATA like-box (ATATTTT) at -26 base pairs (bp), and two CCAAT boxes are positioned at -70 and -100 bp. A 38-bp poly(CA) sequence was located 992 bp upstream from the initiation site. Transient transfection assays in glioblastoma cells and leukemia cells indicate that a 92-bp region was required for basal level PAX6 promoter activity. A negative transcriptional element, silencer (bases -1518 to -1268), functioned differently in different cell lines. The activation of the promoter is positively correlated with the expression of PAX6 transcripts in all cells tested. These results indicate that a cis-regulatory element or elements is responsible for selective activation of the PAX6 promoter in cells that can express PAX6 mRNA.

Base Sequence↗

iMOT: an interactive package for the selection of spatially interacting motifs.

Functional selection and three-dimensional structural constraints of proteins relate to the retention of significant sequence similarity between proteins of similar fold and function despite poor overall sequence identity and evolutionary pressures. We report the availability of 'iMOT' (interacting MOTif) server, an interactive package for the automatic identification of spatially interacting motifs among distantly related proteins sharing similar folds and possessing common ancestral lineage. Spatial interactions between conserved stretches of a protein are evaluated by calculations of pseudo-potentials that describe the strength of interactions. Such an evaluation permits the automatic identification of highly interacting conserved regions of a protein. Interacting motifs have been shown to be useful in searching for distant homologues and establishing remote homologies among the largely unassigned sequences in genome databases. Information on such motifs should also be of value in protein folding, modelling and engineering experiments. The iMOT server can be accessed from http://www.ncbs.res.in/~faculty/mini/imot/iMOTserver.html. Supplementary Material can be accessed from: http://www.ncbs.res.in/~faculty/mini/imot/supplementary.html.

Amino Acid Motifs↗

PoweREST: Statistical Power Estimation for Spatial Transcriptomics Experiments to Detect Differentially Expressed Genes Between Two Conditions.

Recent advancements in Spatial Transcriptomics (ST) have significantly enhanced biological research in various domains. However, the high cost of current ST data generation techniques restricts its application in large-scale population studies. Consequently, there is a pressing need to maximize the use of available resources to achieve robust statistical power. One fundamental question in ST analysis is to detect differentially expressed genes (DEGs) among different conditions using ST data. Such DEG analysis is often performed but the associated power calculation is rarely discussed in the literature. To address this gap, we introduce, PoweREST (https://github.com/lanshui98/PoweREST), a power estimation tool designed to support power calculation of DEG detection with 10X Genomics Visium data. PoweREST enables power estimation both before any ST experiments or after preliminary data are collected, making it suitable for a wide variety of power analyses in ST studies. We also provide a user-friendly, program-free web application (https://lanshui.shinyapps.io/PoweREST/), allowing users to interactively calculate and visualize the study power along with relevant the parameters.

Differentially expressed genes↗

Molecular cloning and transcriptional activation of lysozyme-encoding cDNAs in the mosquito Aedes aegypti.

Lysozymes are enzymes characterized by their ability to break down bacterial cell walls. In insects certain lysozymes are only found in the midgut, whereas others are only found in the haemolymph and fat body after immune challenge. We identified two lysozyme-encoding cDNAs from Aedes aegypti. Both deduced protein sequences are basic in nature, contain 148 amino acids including eight highly conserved cysteine residues, and their genomic sequences contain a single intron. Transcriptional profiles indicated that the predominant form is constitutively expressed and up-regulated upon immune challenge and blood feeding in adult mosquitoes. The second form is expressed during early developmental stages, larvae and pupae, and at low levels in adults after immune challenge. Lysozymes in Aedes aegypti play both roles, defined by the spatial and temporal regulation of their expression.

Aedes↗

Systems biology of virus entry in mammalian cells.

In this article, we define systems biology of virus entry in mammalian cells as the discipline that combines several approaches to comprehensively understand the collective physical behaviour of virus entry routes, and to understand the coordinated operation of the functional modules and molecular machineries that lead to this physical behaviour. Clearly, these are extremely ambitious aims, but recent developments in different life science disciplines slowly allow us to set them as realistic, although very distant, goals. Besides classical approaches to obtain high-resolution information of the molecules, particles and machines involved, we require approaches that can monitor collective behaviour of many molecules, particles and machines simultaneously, in order to reveal design principles of the systems as a whole. Here we will discuss approaches that fall in the latter category, namely time-lapse imaging and single-particle tracking (SPT) combined with computational analysis and modelling, and genome-wide RNA interference approaches to reveal the host components required for virus entry. These techniques should in the future allow us to assign host genes to the systems' functions and characteristics, and allow emergence-driven, in silico assembly of networks that include interactions with increasing hierarchy (molecules-multiprotein complexes-vesicles and organelles), and kinetics and subcellular spatiality, in order to allow realistic simulations of virus entry in real time.

Animals↗

Bipartite structure and functional independence of adenovirus type 5 packaging elements.

Selectivity and polarity of adenovirus type 5 DNA packaging are believed to be directed by an interaction of putative packaging factors with the cis-acting adenovirus packaging domain located within the genomic left end (nucleotides 194 to 380). In previous studies, this packaging domain was mutationally dissected into at least seven functional elements called A repeats. These elements, albeit redundant in function, exhibit differences in the ability to support viral packaging, with elements I, II, V, and VI as the most critical repeats. Viral packaging was shown to be sensitive to spatial changes between individual A repeats. To study the importance of spatial constraints in more detail, we performed site-directed mutagenesis of the 21-bp linker regions separating A repeats I and II, as well as A repeats V and VI. The results of our mutational analysis reveal previously unrecognized sequences that are critical for DNA encapsidation in vivo. On the basis of these results, we present a more complex consensus motif for the adenovirus packaging elements which is bipartite in structure. DNA encapsidation is compromised by changes in spacing between the two conserved parts of the consensus motif, rather than between different A repeats. Genetic evidence implicating packaging elements as independent units in viral DNA packaging is derived from the selection of revertants from a packaging-deficient adenovirus: multimerization of packaging repeats is sufficient for the evolution of packaging-competent viruses. Finally, we identify minimally sized segments of the adenovirus packaging domain that can confer viability and packaging activity to viruses carrying gross truncations within their left-end sequences. Coinfection experiments using the revertant as well as the minimal-packaging-domain mutant viruses strengthen existing arguments for the involvement of limiting, trans-acting components in viral DNA packaging.

Adenoviridae↗

Nuclear organization in differentiating oligodendrocytes.

Many studies have suggested that the 3D organization of chromatin and proteins within the nucleus contributes to the regulation of gene expression. We tested multiple aspects of this nuclear organization model within a primary cell culture system. Oligodendrocyte lineage cells were examined to facilitate analysis of nuclear organization relative to a highly expressed tissue-specific gene, proteolipid protein (PLP), which exhibits transcriptional upregulation during differentiation from the immature progenitor stage to the mature oligodendrocyte stage. Oligodendrocyte lineage cells were isolated from brains of neonatal male rodents, and differentiation from oligodendrocyte progenitors to mature oligodendrocytes was controlled with culture conditions. Genomic in situ hybridization was used to detect the single copy of the X-linked PLP gene within each interphase nucleus. The PLP gene was not randomly distributed within the nucleus, but was consistently associated with the nuclear periphery in both progenitors and differentiated oligodendrocytes. PLP and a second simultaneously upregulated gene, the myelin basic protein (MBP) gene, were spatially separated in both progenitors and differentiated oligodendrocytes. Increased transcriptional activity of the PLP gene in differentiated oligodendrocytes corresponded with local accumulation of SC35 splicing factors. Differentiation did not alter the frequency of association of the PLP gene with domains of myelin transcription factor 1 (Myt1), which binds the PLP promoter. In addition to our specific findings related to the PLP gene, these data obtained from primary oligodendrocyte lineage cells support a nuclear organization model in which (1). nuclear proteins and genes can exhibit specific patterns of distribution within nuclei, and (2). activation of tissue-specific genes is associated with changes in local protein distribution rather than spatial clustering of coordinately regulated genes. This nuclear organization may be critical for complex nucleic-acid-protein interactions controlling normal cell development, and may be an important factor in aberrant regulation of cell differentiation and gene expression in transformed cells.

Active Transport, Cell Nucleus↗

Maturation of pig oocytes in vivo and in vitro.

In this review the concept that the origins of embryonic failure occur during oocyte development is explored. The four factors that determine oocyte viability, namely a normal growth phase, adequate follicle cell support during maturation, the completion of intracellular reprogramming before fertilization and the functioning of oocyte surveillance mechanisms, form the four sections of this review. The viability of pig oocytes at the end of the growth phase is compromised by presumptive spontaneous meiotic progression and by morphological heterogeneity. Determining the percentage and identity of viable dictyate oocytes, and identifying the reasons for the loss of viability, are key areas of future investigation. Although the requirement for follicle cell support during maturation is already established, little is yet known about the underlying signals and their transmission to the oocyte. The analysis of the action and nature of somatic signals will provide the foundation for further advances in the maturation of oocytes in vitro. Signalling cascades in oocytes control both the translation of masked mRNA and the modification and spatial localization of resultant proteins. The interdependent nature of this control system explains why inappropriate signals during maturation lead to subsequent embryonic mortality. Chromosomal errors during meiosis and early mitosis accumulate because of the leaky nature of the checkpoint system during the maternally regulated part of development: effective cell cycle surveillance is established only after the activation of the embryonic genome. In summary, we emphasize that the quality of the dictyate oocyte and the provision of appropriate signals in vitro are the principal determinants of maturational success.

Animals↗

RNF43 Mutations Are Associated With the Classical Molecular Subtype, Vigorous Antitumor Immune Responses, and Prolonged Survival in Pancreatic Adenocarcinoma.

RNF43 mutations were correlated with microsatellite status in colorectal cancer and with fewer and later recurrences in pancreatic ductal adenocarcinoma (PDAC). Here, we undertake a detailed assessment of RNF43 mutations in PDAC. A total of 313 PDACs (308 microsatellite stable [MSS] and 5 microsatellite-instable [MSI] cases) underwent next-generation sequencing (Oncomine Tumor Mutation Load assay; Thermo Fisher). Spatial analyses (NanoString) classified PDACs according to their transcriptomic and proteomic immune signaling. Fluorescent imaging was used to define spatial compartments (tumor: pancytokeratin+/CD45- and leukocytes: pancytokeratin-/CD45+). Each of 20 PDACs with RNF43 mutations (RNF43mut) and without RNF43 mutations (RNF43wt) underwent multiplex immunofluorescence analysis to determine immune status. A total of 153 PDACs (22 RNF43mut and 131 RNF43wt cases) underwent bulk RNA sequencing to assign into molecular subtypes. Overall, 24 RNF43 mutations were identified (22 MSS PDACs and 2 MSI PDACs). The incidence of RNF43 mutations in MSS PDACs (7.1%) was consistent with The Cancer Genome Atlas (6.7%). However, RNF43 mutations were more frequent among MSI PDACs (40%). Additionally, RNF43mut had differential frequencies of other mutations (including Wnt pathway genes), higher tumor mutational burden values (5.5 mut/mb vs 1.67 mut/mb; P < .01), and significantly longer overall survival (47 vs 18 months; P < .0001) than RNF43wt. Moreover, RNF43mut exhibited significantly higher densities of CD8+ T lymphocytes, dendritic cells, and B lymphocytes (P < .001) and an upregulation of ITGAX, CD11c, CD8, and HLA-DR compared with RNF43wt. Patients with RNF43mut PDACs were more often of the classical molecular subtype (20/22, 90.9%). RNF43mut PDACs showed high tumor mutational burden values, suggesting increased neoantigen load coupled with an abundance of antigen-presenting immune cells and an upregulation of immune determinants promoting antigen presentation. All this contributes to stronger antitumor immune responses and improved clinical outcomes.

Humans↗

Studying large viruses.

In this article we have attempted to describe some structural aspects of large viruses. Although this may seem a straightforward task, it is complicated by the fact that large viruses do not represent a distinctive class of organisms and any grouping under this heading will include a range of unrelated viruses with different structures, replication strategies, and host types. To simplify matters we limited our definition to dsDNA viruses with genomes of 100 kbp or larger. However, even this restricted grouping includes viruses with diverse and seemingly unrelated structures. Furthermore, few if any structural features are exclusive to large viruses and most of what appears distinctive about their structure or assembly can also be found in smaller, and usually better characterized, viruses. Therefore we have not attempted to provide a comprehensive catalog of the properties of large viruses but have tried to illustrate particular structural points with examples from a few of the better known forms, notably herpes simplex virus (HSV) and phage T4. The two techniques used to provide rigorous analyses of virus structures are X-ray crystallography and electron cryomicroscopy with computer-assisted reconstruction. To date, X-ray crystallography has been successful only with smaller viruses, and what is known about the structures of these large viruses has come primarily from electron cryomicroscopy. However, with the notable exception of the HSV capsid, such studies have been limited in extent and of relatively low resolution, and the information obtained has been confined largely to describing the spatial distributions and relationships between the subunits. Nevertheless, these studies have given us our clearest insights into the biology of these complex particles and increases in resolution promise to extend these insights by bridging the gap between gross and atomic structures, as exemplified by the identification and mapping of secondary structural elements in the HSV capsid.

Genome, Viral↗

Enzyme-linked fluorescent detection for automated multiplex DNA sequencing.

Initiatives to sequence DNA on a large scale have created a need for increased throughput and decreased costs. One scheme for increasing throughput, multiplex sequencing, involves the processing of a mixture of sequencing templates followed by sequential hybridization to reveal the individual sequence ladders on a membrane. Because multiplex sequencing has not been fully automated, and has not seemed automatable, few sequencing efforts have attempted to exploit it. We describe here a scheme for the automation of multiplex sequencing. Probe hybridized to target DNA is detected via spatially localized enzyme-linked fluorescence. Light output is high enough that imaging is possible with simple instrumentation. Direct imaging within an automated hybridization apparatus is made feasible so that the entire process will be automatic once a multiplex membrane is produced. The technique has the potential to increase severalfold the throughput of automated sequencing instruments required for sequencing the human genome.

Base Sequence↗

Regulation of the human poly(ADP-ribosyl) transferase promoter via alternative DNA racket structures.

Human nuclear poly(ADP-ribosyl) transferase (ADPRT) protein content in cells suggests that ADPRT expression is stringently controlled. Analysis of the 3 kb promoter sequence, which is required for high level expression, revealed an extraordinary architecture: several Sp1 motifs are located in the vicinity of the first exon but the closest CCAAT/TATA boxes are several hundred basepairs away. Four Alu type repetitive sequences are in the promoter structure. Within these Alu sequences there exist inverted repeat elements, which could form two mutually exclusive types of DNA tertiary structure consisting of quadruplex DNA and loops resembling rackets. Thereby, a CCAAT/TATA element would be moved to spatial vicinity of the Sp1 site activating the promoter. Deletion analysis showed the functional significance of these racket elements. We also obtained evidence for DNA racket structures when we studied mutational mechanisms in a human adenine phosphoribosyltransferase (APRT) deficient patient. One of his alleles harbours a novel complex type of deletion/insertion mutation. Based on several highly informative sequence features in this genomic region a model is proposed for the generation of this unusual type of mutation involving two steps: an initial targeting step and a subsequent complex rearrangement. This process includes the formation of a DNA racket structure, which resembles that of the ADPRT promoter. Thus we conclude that DNA racket structures seem to be of general importance in nature.

Base Sequence↗

Comprehensive analysis of combinatorial regulation using the transcriptional regulatory network of yeast.

Studies on various model systems have shown that a relatively small number of transcription factors can set up strikingly complex spatial and temporal patterns of gene expression. This is achieved mainly by means of combinatorial or differential gene regulation, i.e. regulation of a gene by two or more transcription factors simultaneously or under different conditions. While a number of specific molecular details of the mechanisms of combinatorial regulation have emerged, our understanding of the general principles of combinatorial regulation on a genomic scale is still limited. In this work, we approach this problem by using the largest assembled transcriptional regulatory network for yeast. A specific network transformation procedure was used to obtain the co-regulatory network describing the set of all significant associations among transcription factors in regulating common target genes. Analysis of the global properties of the co-regulatory network suggested the presence of two classes of regulatory hubs: (i) those that make many co-regulatory associations, thus serving as integrators of disparate cellular processes; and (ii) those that make few co-regulatory associations, and thereby specifically regulate one or a few major cellular processes. Investigation of the local structure of the co-regulatory network revealed a significantly higher than expected modular organization, which might have emerged as a result of selection by functional constraints. These constraints probably emerge from the need for extensive modular backup and the requirement to integrate transcriptional inputs of multiple distinct functional systems. We then explored the transcriptional control of three major regulatory systems (ubiquitin signaling, protein kinase and transcriptional regulation systems) to understand specific aspects of their upstream control. As a result, we observed that ubiquitin E3 ligases are regulated primarily by unique transcription factors, whereas E1 and E2 enzymes share common transcription factors to a much greater extent. This suggested that the deployment of E3s unique to specific functional contexts may be mediated significantly at the transcriptional level. Likewise, we were able to uncover evidence for much higher upstream transcription control of transcription factors themselves, in comparison to components of other regulatory systems. We believe that the results presented here might provide a framework for testing the role of co-regulatory associations in eukaryotic transcriptional control.

Algorithms↗

Comprehensive Transcriptome Annotation of Thousands of HIV-1 Genomes.

Alternative splicing in HIV-1 has been a central focus of decades of research, uncovering key mechanisms of viral gene regulation, immune evasion, and therapeutic response - yet, no reference resource has existed to support transcriptome-wide analysis, limiting adoption of modern computational methods. We present HIV Atlas (https://ccb.jhu.edu/HIV_Atlas), the first reference-quality annotation of HIV-1 and SIV transcriptional diversity. We manually curated transcriptomes for HIV-1HXB2 and SIVmac239 and developed Vira, an automated annotation-transfer method specifically designed to address unique challenges of viral genome biology, to generate high-quality annotations for 2,077 complete HIV-1 genomes. Using the resources presented in our work, we evaluated conservation of splice sites, revealing near-perfect preservation of major donors and acceptors. Furthermore, using several public datasets, we demonstrate how HIV Atlas enhances methodology, improves the quality and novelty of results, and opens novel avenues for research, supporting more accurate and comprehensive analyses of bulk, single-cell, and spatial RNA-seq in HIV-1 studies.

Journal Article↗

Drosophila as a model system for molecular analysis of tumorigenesis.

In Drosophila, homozygous mutations in a series of genes can cause the appearance of tissue-specific tumors. These tumors occur either during embryonic or larval development. The majority of the identified genes give rise to larval tumors that affect either the presumptive adult optic centers of the brain, the imaginal discs, the hematopoietic organs, or the germ cells. These genes act as recessive determinants of neoplasia and have been designated as tumor-suppressor genes. They are normally required for the regulation of cell proliferation and cell differentiation during development. Among these genes, the lethal(2)giant larvae (l(2)gl) has been best studied. Homozygous mutations in l(2)gl produce malignant tumors in the brain hemispheres and the imaginal discs. The l(2)gl gene has been cloned, introduced back into the genome of l(2)gl-deficient animals, and shown to restore normal development. The nucleotide sequence of the l(2)gl gene has been determined, as well as the sequence of its transcripts. Anti-l(2)gl antibodies recognize a protein of about 130 kDa that corresponds to the major product of l(2)gl transcripts. Analysis of the spatial distribution of l(2)gl transcripts and proteins revealed a first phase of intensive expression during embryogenesis and a second weaker phase during the larval to pupal transition period. As revealed by mosaic experiments, the critical period of l(2)gl expression for preventing tumorigenesis takes place during early embryogenesis. During this period, the l(2)gl protein is ubiquitously expressed in all cells and tissues, while during late embryogenesis expression becomes gradually restricted to the midgut epithelium and the axon projections of the ventral nervous system that show no phenotypic alteration in the mutant animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Spatial-temporal and phylogenetic analyses of epidemiologic data to help understand the modes of transmission of endemic typhoid fever in Samoa.

Salmonella enterica serovar Typhi (S. Typhi) is either widely distributed or proximally transmitted via fecally-contaminated food or water to cause typhoid fever. In Samoa, where endemic typhoid fever has persisted over decades despite water quality and sanitation improvements, the local patterns of S. Typhi circulation remain unclear. From April 2018-June 2020, epidemiologic data and GPS coordinates were collected during household investigations of 260 acute cases of typhoid fever, and 27 asymptomatic shedders of S. Typhi were detected among household contacts. Spatial and temporal distributions of cases were examined using Average Nearest Neighbor and space-time hotspot analyses. In rural regions, infections occurred in sporadic, focal clusters contrasting with persistent, less clustered cases in the Apia Urban Area. Restrictions to population movement during nationwide lockdowns in 2019-2020 were associated with marked reductions of cases. Phylogenetic analyses of isolates with whole genome sequences (n = 186) revealed one dominant genotype 3.5.4 (n = 181/186) that contains three Samoa-exclusive sub-lineages: 3.5.4.1, 3.5.4.2, and 3.5.4.3. Variables of patient sex, age, and geographic region were examined by phylogenetic groupings, and significant differences (p<0.05) associated genetically-similar isolates in urban areas with working ages (20-49 year olds), and in rural areas with age groups typically at home (<5, 50+). Isolates from asymptomatic shedders were among all three sub-lineages. Whole genome sequencing provided evidence of bacterial genetic similarity, which corroborated 10/12 putative epidemiologic linkages among cases and asymptomatic shedders, as well as 3/3 repeat positives (presumed relapses), with a median of one single nucleotide polymorphism difference. These findings highlight various patterns of typhoid transmission in Samoa that differ between urban and rural regions as well as genomic subtypes. Asymptomatic shedders, detectable only through household investigations, are likely an important reservoir and mobile agent of infection. This study advances a "Samoan S. Typhi framework" that supports current and future typhoid surveillance and control efforts in Samoa.

Humans↗

Molecular characterization of a deletion mutation affecting the alpha'-subunit of beta-conglycinin of soybean.

A naturally occurring variety of soybean Glycine max cv. Keburi, which lacks the alpha'-subunits of the 7S seed storage protein (beta-conglycinin), was recently described by Kitamura and Kaizuma. Keburi beta-conglycinins contain a normal complement of alpha-, beta-, and gamma-subunits that accumulate in a temporally and spatially regulated manner comparable to that of the standard cultivar Provar. Poly(A)+ RNA isolated from mid-to-late maturation stage Keburi seeds was translated in vitro, yielding products equivalent to those of Provar poly(A)+ RNA except that Keburi mRNA did not produce the pre-alpha'-subunit polypeptide. The basis of the Keburi phenotype was determined by examining genomic DNA using Southern blot hybridization. Restriction fragments isolated from a cloned 11.5 kb EcoRI fragment of genomic DNA containing a normal alpha'-subunit gene (Gmg 17.1) were used as hybridization probes. Sequences far upstream of the alpha'-subunit gene were present in both Provar and Keburi cultivars. However, hybridization reactions with probes from within the gene demonstrated that a deletion had occurred in Keburi DNA beginning immediately 5' to the alpha'-subunit gene represented on this 11.5 kb fragment. The deletion extends through most of the coding sequences, producing the Keburi phenotype.

Antigens, Plant↗

Genetically variable nucleopolyhedroviruses isolated from spatially separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) in Orkney.

Here we report a lepidopteran system in which a pathogen is both abundant and genotypically variable. Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles to investigate the prevalence of a pathogen, O. brumata Nucleopolyhedrovirus (OpbuNPV), within the natural system. Virus was recorded in 11 of the 13 winter moth populations sampled, with two populations suffering mortality due to virus at levels of 50%. The virus genome from 200 single insect isolations was investigated for variation using restriction endonuclease digests. Twenty-six variants of OpbuNPV were detected using SalI. The polyhedrin gene of the virus was partially sequenced, allowing the relationship between the 26 variants to be portrayed as a cladogram. The phylogenetic relationship between OpbuNPV and other known baculovirus polyhedrin gene sequences was also established. The discovery of virus at such high prevalence is discussed with reference to occurrence and genetic variation of pathogens in other lepidopteran host populations. This study shows encouraging results for further studies into the role of pathogens in the regulation of host insect populations.

Animals↗