Gastric Cecum Research Articles

Targeting Syntaxin1A via RNA interference inhibits feeding and midgut development in Locusta migratoria.

Syntaxin1A (Syx1A) has diverse and indispensable functions in animals. Previous studies have mainly focused on the roles of Syx1A in Drosophila, and so how Syx1A operates during the development of other insects remains poorly understood. This study investigated whether disrupting LmSyx1A using RNA interference (RNAi) affects the growth and development of Locusta migratoria. LmSyx1A was expressed in all tissues tested, with the highest expression observed in the fat body. After 5th-instar nymphs were injected with double-stranded LmSyx1A (dsLmSyx1A), none of the nymphs were able to molt normally and all eventually died. The silencing of LmSyx1A resulted in the cessation of feeding, body weight loss, and atrophy of the midgut and gastric cecum in locusts. Hematoxylin and eosin (H&E) staining showed that the columnar cells in the midgut were severely damaged, with microvilli defects visible in dsLmSyx1A-injected nymphs. Secretory vesicles were observed with transmission electron microscopy (TEM). In addition, reverse transcription quantitative polymerase chain reaction (RT-qPCR) further indicates that silencing LmSyx1A repressed the expression of genes involved in the insulin/mammalian target of rapamycin (mTOR)-associated nutritional pathway. Taken together, these results suggest that LmSyx1A significantly affects the midgut cell layer of locust nymphs, which was partially associated with the downregulation of the insulin/mTOR-associated nutritional pathway. Thus, we argue that LmSyx1A is a suitable target for developing dsRNA-based biological pesticides for managing L.migratoria.

Insecticidal Activity and Molecular Target by Morphological Analysis, RNAseq, and Molecular Docking of the Aryltetralin Lignan Lactone Helioxanthin, Isolated from Taiwania flousiana Gaussen.

Botanical insecticides are considered an environmentally friendly approach to insect control because they are easily biodegraded and cause less environmental pollution compared to traditional chemical pesticides. In this study, we reported the insecticidal activities of the ingredients from Taiwania flousiana Gaussen (T. flousiana). Five compounds, namely helioxanthin (C1), taiwanin E (C2), taiwanin H (C3), 7,4'-dimethylamentoflavone (C4), and 7,7″-di-O-methylamentoflavone (C5), were isolated and tested against the second, third, and fourth instar larvae of Aedes aegypti. Our results indicated that all five compounds showed insecticidal activities, and helioxanthin, which is an aryltetralin lignan lactone, was the most effective with LC50 values of 0.60, 2.82, and 3.12 mg/L, respectively, 48 h after application, with its activity against the second instar larvae similar to that of pyrethrin and better than that of rotenone. Further studies found that helioxanthin accumulated in the gastric cecum and the midgut and caused swelling of mitochondria with shallow matrices and fewer or disappeared crista. Additionally, our molecular mechanisms studies indicated that the significantly differentially expressed genes (DEGs) were mainly associated with mitochondria and the cuticle, among which the voltage-dependent anion-selective channel (VDAC) gene was the most down-regulated by helioxanthin, and VDAC is the potential target of helioxanthin by binding to specific amino acid residues (His 122 and Glu 147) via hydrogen bonds. We conclude that aryltetralin lignan lactone is a potential class of novel insecticides by targeting VDAC.

A quantitative micro-tomographic gut atlas of the lepidopteran model insect Manduca sexta

The tobacco hornworm is used extensively as a model system for ecotoxicology, immunology and gut physiology. Here, we established a micro-computed tomography approach based on the oral application of the clinical contrast agent iodixanol, allowing for a high-resolution quantitative analysis of the Manduca sexta gut. This technique permitted the identification of previously unknown and understudied structures, such as the crop or gastric ceca, and revealed the underlying complexity of the hindgut folding pattern, which is involved in fecal pellet formation. The acquired data enabled the volume rendering of all gut parts, the reliable calculation of their volumes, and the virtual endoscopy of the entire alimentary tract. It can provide information for accurate orientation in histology uses, enable quantitative anatomical phenotyping in three dimensions, and allow the calculation of locally effective midgut concentrations of applied chemicals. This atlas will provide critical insights into the evolution of the alimentary tract in lepidopterans.

Morphological Study of the Alimentary Canal and Malpighian Tubules in the Adult of the Pollen Beetle Meligethes (Odonthogethes) chinensis (Coleoptera: Nitidulidae: Meligethinae)

Meligethes (Odonthogethes) chinensis is a highly specialized species of Nitidulidae in China that takes pollen as its main food source, and its main host plant is Rubus idaeus L. (Rosaceae). In this study, the structural morphology of the alimentary canal and Malpighian tubules of adult M. (O.) chinensis was observed under light, fluorescence, and scanning electron microscopy. The alimentary canal of adult M. (O.) chinensis is divided into foregut, midgut, and hindgut. The foregut is the shortest and consists of the pharynx, esophagus, proventriculus, and cardiac valve. The midgut is a straight, distended, cylindrical, thin-walled tube. Numerous blunt-fingered gastric ceca are distributed irregularly throughout the midgut. The hindgut is subdivided into the ileum, colon, and rectum. The ileum is coiled. The colon gradually enlarges posteriorly. The rectum is thickly muscled and followed by a membranous structure. The openings of proximal Malpighian tubules are evenly inserted into the junction of the midgut and hindgut, and distal Malpighian tubules are evenly attached to the colon to form a cryptonephridial system. In this study, we also compare the structure and infer the function of the alimentary canal and Malpighian tubules among beetles, as well as discuss the evolutionary and taxonomical implications.

Pathology of an iridescent virus in immature Culex pipiens L. (Diptera, Culicidae).

Iridovirus in Culex pipiens was reported for the first time in 2012. Later studies of horizontal transmission were performed, in which an interaction with the parasite Strelkovimermis spiculatus acting as viral vector was recognized. In the present study, we observed aspects of the pathology produced by an invertebrate iridescent virus in laboratory infected immature Cx. pipiens as well as in infected immature Cx. pipiens in the field. In the laboratory infected larvae, the infection and mortality were asynchronous. Signs of infection in larvae exposed to the virus were observed between the second and the fourth days post-exposure in 99% of the cases, while the highest daily record of visible infected larvae (52%) was observed on the third day post exposure. Moreover, 79% of confirmed virus infected larvae died in the first 10 days after exposure. The Median Lethal Time was eight days. Several tissues were found to be infected and the common sites of replication were the fat body, epidermis and epithelial derivatives, such as the imaginal discs and the tracheal epithelium. Moreover, infection in the salivary glands, gastric ceca and posterior gut have not been previously documented on other mosquito iridescent viruses.

DEAD-Box RNA Helicase DDX47 Maintains Midgut Homeostasis in Locusta migratoria.

Tissue homeostasis is critical for maintaining organ shape, size, and function. The condition is regulated by the balance between the generation of new cells and the loss of senescent cells, and it involves many factors and mechanisms. The midgut, an important part of the intestinal tract, is responsible for digestion and nutrient absorption in insects. LmDDX47, the ortholog of DEAD-box helicase 47 from Locusta migratoria, is indispensable for sustaining a normal midgut in the nymphs. However, the underlying cellular and molecular mechanisms remain to be elucidated. In this study, LmDDX47 knockdown resulted in atrophy of the midgut and gastric cecum in both nymph and adult locusts. After LmDDX47 knockdown, the number of regenerative and columnar cells in the midgut was significantly reduced, and cell death was induced in columnar tissue. LmDDX47 was localized to the nucleolus; this was consistent with the reduction in 18S rRNA synthesis in the LmDDX47 knockdown group. In addition, the acetylation and crotonylation levels of midgut proteins were significantly increased. Therefore, LmDDX47 could be a key regulator of midgut homeostasis, regulating 18S rRNA synthesis as well as protein acetylation and crotonylation in the migratory locust.

Genomic and expression characterization of aquaporin genes from Siniperca chuatsi

Aquaporins (AQPs) are major intrinsic proteins that form pores in the membranes of biological cells. We first cloned the full-length sequences of aqp0, 1, 3, 4, 7, 8, 9, 10, 11, and 12 genes in Siniperca chuatsi. The 10 S. chuatsi aqp (Sc-aqp) genes included complete open reading frames and exhibited different exon-intron organizations. Sc-aqp1, 3, 8, 9, 10, and 11 were mostly expressed in the gallbladder, gills, gastric cecum, liver, ovaries, and spleen, respectively; Sc-aqp0 and 4 were mostly expressed in larvae at 1 day after hatching and in gastrula; Sc-aqp7 and 12 were mostly expressed in 2K-cell embryos. The expression levels of Sc-aqp1, 3, 7, 8, 9, and 10 after 10 part per thousand (ppt) salt treatment had significantly changed compared with those after 0 ppt salt treatment. Real-time quantitative PCR analysis further showed that in the intestines, the mRNA levels of Sc-aqp1 and 10 significantly decreased by approximately 2.07- and 2.85-fold, respectively, whereas those of Sc-aqp8 and 9 significantly increased by approximately 7.08- and 4.14-fold, respectively. Sc-aqp1, 8, 9, and 10 showed no significant differences in the gills. Sc-aqp3 significantly decreased by approximately 1.51- and 1.67-fold in the gills and intestines, respectively. Sc-aqp7 significantly increased by approximately 4.18- and 7.04-fold in the gills and intestines, respectively. This study was the first to investigate the tissue expression profiles and response to salt stress of aqp genes in S. chuatsi. Moreover, altering diet and suffering from immune stress could cause changes in the expression level of aqps. This study provided valuable reference information for AQPs' roles in osmoregulation in freshwater fish.

Sequence and functional analysis of intestinal alkaline phosphatase from Lateolabrax maculatus.

Alkaline phosphatases (Alps) belong to a class of phosphate transferases that dephosphorylate lipopolysaccharide (LPS), adenosine triphosphate, and nucleotides. In this study, a 1874-base pair (bp) intestinal alp cDNA sequence was cloned from Lateolabrax maculatus and designated as Lm-alpi. It contained a 1611bp open reading frame which encoded a protein with 537 amino acids. Protein sequence alignment showed that Lm-AlpI shared 29.8-79.8% identity with its homologs. Lm-AlpI catalytic sites contained three metal ion sites (two Zn2+ and one Mg2+), referring to D73, H184, D348, H349, H352, H464, D389, and H390 residues, which are essential for enzymatic activity and conservation in different organisms. Two predicted disulfide bonds in Lm-AlpI were composed of four cysteines (C152-C214 and C499-C506), which were homologous to those of mammals. Immunohistochemical staining revealed that Lm-AlpI was mainly expressed on the mucosal surface of the gastrointestinal tract, including stomach, intestine, and gastric cecum. Lm-AlpI was mainly located on the plasma membrane of transiently transfected HeLa cells. The mRNA of Lm-alpi was mainly expressed in the intestine, and its expression levels gradually increased after LPS treatment and further increased by 1.81-fold after 48h. After desalting culture, the relative mRNA expression level of Lm-alpi decreased at 30 and 50days after hatching (DAH) and then returned to normal levels at 70 DAH. Further experiments demonstrated that the enzyme activity of Lm-AlpI exhibited an expression pattern similar to that of the mRNA expression of Lm-alpi after LPS treatment and desalting culture. This study provided valuable information on the Lm-AlpI functions associated with the mucosal immunity and salinity adaptation of L. maculatus.

Study of histoarchitectural changes in Anopheles stephensi larvae following exposure to Eucalyptus globulus and Aloe vera oils

Chemical-based insecticides/larvicides pose a great threat to humans. The present study describes the histopathological changes in Anopheles stephensi Liston (Diptera: Culicidae) larvae after exposure to two essential oils, i.e. Eucalyptus globulus and Aloe vera. The exposure of 4th instar larvae of An. stephensi to 90 ppm of E. globulus and 550 ppm of A. vera oils resulted in various histological alterations in organs of the head regions like disintegration of the brain and other structures, disorganization in the imaginal buds of the antennae, complete disappearance of the optic lobes, and total disruption of the inner and outer retractor muscles of the brush as compared to the control larvae. Oil-exposed larvae also exhibited diversifications in gastric ceca, disintegration of the midgut epithelium layer, rifts in microvilli, and disappearance or reduction of fat bodies as well as the epithelium layer of the hindgut. Such pathological changes restrict the larvae's ability to perform various functions, leading to their death.

Identification and functional analysis of a cytochrome P450 gene CYP9AQ2 involved in deltamethrin detoxification from Locusta migratoria

A 1578-bp cDNA of a cytochrome P450 gene (CYP9AQ2) was sequenced from the migratory locust, Locusta migratoria. It contains an open reading frame (ORF) of 1557 bp that encodes 519 amino acid residues. As compared with other known insect cytochrome P450 enzymes, the overall structure of its deduced protein is highly conserved. The expression of CYP9AQ2 was relatively higher in nymphal stages than in egg and adult stages, and the highest expression was found in fourth-instar nymphs, which was 8.7-fold higher than that of eggs. High expression of CYP9AQ2 was observed in foregut, followed by hindgut, Malpighian tubules, brain and fat bodies, which were 75~142-fold higher than that in hemolymph. Low expression was found in midgut, gastric cecum and hemolymph. The expression of CYP9AQ2 was up-regulated by deltamethrin at the concentrations of 0.04, 0.08, and 0.12 µg/mL and the maximal up-regulation was 2.6-fold at LD10 (0.04 µg/mL). RNA interference-mediated silencing of CYP9AQ2 led to an increased mortality of 25.3% when the nymphs were exposed to deltamethrin, suggesting that CYP9AQ2 plays an important role in deltamethrin detoxification in L. migratoria. Computational docking studies suggested that hydroxylation of the phenoxybenzyl moiety might be one of the deltamethrin metabolic pathways by CYP9AQ2.

Habitat visualization and genomic analysis of "Candidatus Pantoea carbekii," the primary symbiont of the brown marmorated stink bug.

Phytophagous pentatomid insects can negatively impact agricultural productivity and the brown marmorated stink bug (Halyomorpha halys) is an emerging invasive pest responsible for damage to many fruit crops and ornamental plants in North America. Many phytophagous stink bugs, including H. halys, harbor gammaproteobacterial symbionts that likely contribute to host development, and characterization of symbiont transmission/acquisition and their contribution to host fitness may offer alternative strategies for managing pest species. “Candidatus Pantoea carbekii” is the primary occupant of gastric ceca lumina flanking the distal midgut of H. halys insects and it is acquired each generation when nymphs feed on maternal extrachorion secretions following hatching. Insects prevented from symbiont uptake exhibit developmental delays and aberrant behaviors. To infer contributions of Ca. P. carbekii to H. halys, the complete genome was sequenced and annotated from a North American H. halys population. Overall, the Ca. P. carbekii genome is nearly one-fourth (1.2 Mb) that of free-living congenerics, and retains genes encoding many functions that are potentially host-supportive. Gene content reflects patterns of gene loss/retention typical of intracellular mutualists of plant-feeding insects. Electron and fluorescence in situ microscopic imaging of H. halys egg surfaces revealed that maternal extrachorion secretions were populated with Ca. P. carbekii cells. The reported findings detail a transgenerational mode of symbiont transmission distinct from that observed for intracellular insect mutualists and illustrate the potential additive functions contributed by the bacterial symbiont to this important agricultural pest.

B chromosomes showing active ribosomal RNA genes contribute insignificant amounts of rRNA in the grasshopper Eyprepocnemis plorans.

The genetic inertness of supernumerary (B) chromosomes has recently been called into question after finding several cases of gene activity on them. The grasshopper Eyprepocnemis plorans harbors B chromosomes containing large amounts of ribosomal DNA (rDNA) units, some of which are eventually active, but the amount of rRNA transcripts contributed by B chromosomes, compared to those of the standard (A) chromosomes, is unknown. Here, we address this question by means of quantitative PCR (qPCR) for two different ITS2 amplicons, one coming from rDNA units located in both A and B chromosomes (ITS2(A+B)) and the other being specific to B chromosomes (ITS2(B)). We analyzed six body parts in nine males showing rDNA expression in their B chromosomes in the testis. Amplification of the ITS2(B) amplicon was successful in RNA extracted from all six body parts analyzed, but showed relative quantification (RQ) values four orders of magnitude lower than those obtained for the ITS(A+B) amplicon. RQ values differed significantly between body parts for the two amplicons, with testis, accessory gland and wing muscle showing threefold higher values than head, gastric cecum and hind leg. We conclude that the level of B-specific rDNA expression is extremely low even in individuals where B chromosome rDNA is not completely silenced. Bearing in mind that B chromosomes carry the largest rDNA cluster in the E. plorans genome, we also infer that the relative contribution of B chromosome rRNA genes to ribosome biogenesis is insignificant, at least in the body parts analyzed.

Systemic Spread and Propagation of a Plant-Pathogenic Virus in European Honeybees, Apis mellifera

ABSTRACTEmerging and reemerging diseases that result from pathogen host shifts are a threat to the health of humans and their domesticates. RNA viruses have extremely high mutation rates and thus represent a significant source of these infectious diseases. In the present study, we showed that a plant-pathogenic RNA virus, tobacco ringspot virus (TRSV), could replicate and produce virions in honeybees, Apis mellifera, resulting in infections that were found throughout the entire body. Additionally, we showed that TRSV-infected individuals were continually present in some monitored colonies. While intracellular life cycle, species-level genetic variation, and pathogenesis of the virus in honeybee hosts remain to be determined, the increasing prevalence of TRSV in conjunction with other bee viruses from spring toward winter in infected colonies was associated with gradual decline of host populations and winter colony collapse, suggesting the negative impact of the virus on colony survival. Furthermore, we showed that TRSV was also found in ectoparasitic Varroa mites that feed on bee hemolymph, but in those instances the virus was restricted to the gastric cecum of Varroa mites, suggesting that Varroa mites may facilitate the spread of TRSV in bees but do not experience systemic invasion. Finally, our phylogenetic analysis revealed that TRSV isolates from bees, bee pollen, and Varroa mites clustered together, forming a monophyletic clade. The tree topology indicated that the TRSVs from arthropod hosts shared a common ancestor with those from plant hosts and subsequently evolved as a distinct lineage after transkingdom host alteration. This study represents a unique example of viruses with host ranges spanning both the plant and animal kingdoms.

Immunohistochemical observations of methionine-enkephalin and delta opioid receptor in the digestive system of Octopus ocellatus

The study was designed to determine whether methionine-enkephalin (met-Enk) or delta opioid receptor was present in the digestive system of Octopus ocellatus. The results showed that they were both in the bulbus oris, esophagus, crop, stomach, gastric cecum, intestine, posterior salivary glands of O. ocellatus, one of them, met-Enk in the rectum, anterior salivary glands, digestive gland. And the distributions were extensive in the digestive system. Strong or general met-Enk immunoreactivity was observed in the inner epithelial cells of the bulbus oris, esophagus, stomach, gastric cecum, intestine, anterior salivary glands and the adventitia of the intestine and rectum, and so was the delta opioid receptor immunoreactivity in the inner epithelial cells of the bulbus oris, esophagus, and crop, however, they were weak in other parts. Combining with delta opioid receptor, met-Enk may be involved in the regulations of food intake, absorption, movement of gastrointestinal smooth muscle and secretion of digestive gland. The different densities of met-Enk and delta opioid receptor may be related to the different functions in the digestive system of O. ocellatus.

Distribution of pepsinogen- and ghrelin-producing cells in the digestive tract of Japanese eel (Anguilla japonica) during metamorphosis and the adult stage

Pepsinogen is the precursor form of the gastric-specific digestive enzyme, pepsin. Ghrelin is a representative gastric hormone with multiple functions in vertebrates, including the regulation of growth hormone release, stimulation of food intake and gastrointestinal motility function. We investigated chronological changes in the distribution of pepsinogen-expressing cells by in situ hybridization and ghrelin-immunoreactive cells by immunohistochemistry in the Japanese eel (Anguilla japonica) during metamorphosis from the leptocephalus sage to the elver stage. The ghrelin-producing cells first appeared in the gastric cecum and pyloric portion of the stomach in the late phase of metamorphosing leptocephali, whereas the pepsinogen-producing cells were first detected in the early phase of the glass-eel stage. These suggest that endocrine cells differentiated earlier than exocrine cells in the eel stomach. Accompanying eel development, the distribution of ghrelin-producing cells spread to the esophagus and other regions of the stomach, but not to the intestine. These results may be related to the changes in dietary habits during metamorphosis in the Japanese eel.

Drosophila FoxO Regulates Organism Size and Stress Resistance through an Adenylate Cyclase

Forkhead box class O (FoxO) transcription factors are a family of conserved proteins that regulate the cellular responses to various stimuli, such as energy deprivation, stress, and developmental cues. FoxO proteins are important mediators of the insulin signaling pathway, adjusting growth and metabolism to nutrient availability. Insulin signaling acts together with the glucagon-stimulated cAMP signaling pathway to orchestrate the organism response to various nutritional conditions. In this study, we demonstrate that Drosophila melanogaster FoxO (dFoxO) regulates cAMP signaling by directly inducing the expression of an adenylate cyclase gene, ac76e. Interestingly, ac76e is expressed in a highly restricted pattern throughout fly development, limited to the corpus allatum (CA), gastric cecum, and malpighian tubules. dFoxO activation of AC76E in the CA increases starvation resistance and limits growth. Our results unravel a new role for dFoxO, integrating cAMP and insulin signaling to adapt organism growth to the existing nutritional conditions.

The olive fly endosymbiont, "Candidatus Erwinia dacicola," switches from an intracellular existence to an extracellular existence during host insect development.

As polyphagous, holometabolous insects, tephritid fruit flies (Diptera: Tephritidae) provide a unique habitat for endosymbiotic bacteria, especially those microbes associated with the digestive system. Here we examine the endosymbiont of the olive fly [Bactrocera oleae (Rossi) (Diptera: Tephritidae)], a tephritid of great economic importance. "Candidatus Erwinia dacicola" was found in the digestive systems of all life stages of wild olive flies from the southwestern United States. PCR and microscopy demonstrated that "Ca. Erwinia dacicola" resided intracellularly in the gastric ceca of the larval midgut but extracellularly in the lumen of the foregut and ovipositor diverticulum of adult flies. "Ca. Erwinia dacicola" is one of the few nonpathogenic endosymbionts that transitions between intracellular and extracellular lifestyles during specific stages of the host's life cycle. Another unique feature of the olive fly endosymbiont is that unlike obligate endosymbionts of monophagous insects, "Ca. Erwinia dacicola" has a G+C nucleotide composition similar to those of closely related plant-pathogenic and free-living bacteria. These two characteristics of "Ca. Erwinia dacicola," the ability to transition between intracellular and extracellular lifestyles and a G+C nucleotide composition similar to those of free-living relatives, may facilitate survival in a changing environment during the development of a polyphagous, holometabolous host. We propose that insect-bacterial symbioses should be classified based on the environment that the host provides to the endosymbiont (the endosymbiont environment).

Co-induction of hepatic IGF-I and progranulin mRNA by growth hormone in tilapia, Oreochromis mossambiccus

Like IGF-I, progranulin (pgrn) is a growth factor involved in tumorigenesis and wound healing. We report here the identification and characterization of pgrn cDNA in tilapia and the regulation of its expression by growth hormone (GH). The tilapia pgrn cDNA was cloned by RT-PCR amplification, using gene specific oligonucleotides as amplification primers. The cDNA contains an open reading frame encoding a peptide of 206 amino acid residues (aa) that contains a presumptive signal peptide (23 aa) and two repeat units of granulin (grn, 51 and 52 aa, respectively) franked by a GAP of 49 aa and the carboxyl terminus with 31 aa. The two predicted grn peptides are arranged in tandem repeats interrupted by a GAP peptide. RT-PCR analysis revealed that high levels of prgn mRNA were present in several tissues such as spleen, gastric cecum, intestine, fat tissue, gill, kidney, eye and pancreas, and lower levels in liver, muscle, heart, brain, skin and stomach. Administration of a single dose (500 ng/g body weight) of recombinant seabream growth hormone (rbGH) by intraperitoneal (ip) injection into one-month-old tilapia resulted in an obvious increase of IGF-I and pgrn mRNA (2.7-fold and 2.5-fold, respectively) in the liver at three hours post-GH treatment. The peptide levels of pgrn in the liver of GH-treated fish also were substantially induced over controls at 12 h post-GH treatment as detected by western immuno-blot analysis. The co-induction of IGF-I and pgrn following GH treatment may suggest the involvement of pgrn in GH regulated growth in tilapia.

Morphological and Molecular Characterization of a Cypovirus ( Reoviridae ) from the Mosquito Uranotaenia sapphirina (Diptera: Culicidae)

A novel cypovirus has been isolated from the mosquito Uranotaenia sapphirina (UsCPV) and shown to cause a chronic infection confined to the cytoplasm of epithelial cells of the gastric ceca and posterior stomach. The production of large numbers of virions and inclusion bodies and their arrangement into paracrystalline arrays gives the gut of infected insects a distinctive blue iridescence. The virions, which were examined by electron microscopy, are icosahedral (55 to 65 nm in diameter) with a central core that is surrounded by a single capsid layer. They are usually packaged individually within cubic inclusion bodies (polyhedra, approximately 100 nm across), although two to eight virus particles were sometimes occluded together. The virus was experimentally transmitted per os to several mosquito species. The transmission rate was enhanced by the presence of magnesium ions but was inhibited by calcium ions. Most of the infected larvae survived to adulthood, and the adults retained the infection. Electrophoretic analysis of the UsCPV genome segments (using 1% agarose gels) generated a migration pattern (electropherotype) that is different from those of the 16 Cypovirus species already recognized. UsCPV genome segment 10 (Seg-10) showed no significant nucleotide sequence similarity to the corresponding segment of the other cypoviruses that have previously been analyzed, and it has different "conserved" termini. A BLAST search of the UsCPV deduced amino acid sequence also showed little similarity to Antheraea mylitta CPV-4 (67 of 290 [23%]) or Choristoneura fumiferana CPV-16 (33 of 111 [29%]). We conclude that UsCPV should be recognized as a member of a new Cypovirus species (Cypovirus 17, strain UsCPV-17).

Observations on the Leech Placobdella ornata Feeding From Bony Tissues of Turtles

The leech Placobdella ornata was observed feeding from the blood sinuses of the plastron and carapace bones of Chelydra serpentina and Chrysemys picta. Evidence of successful feeding included blood upwelling from the point of attachment and gastric ceca of the leeches freshly filled with blood after removal. There was an apparent preference for the sulci between scales of the shell.