Differentiation of three species of ixodid tick, Dermacentor andersoni, D. variabilis and D. albipictus, by PCR-based approaches using markers in ribosomal DNA

Three species of Dermacentor, Dermacentor albipictus, Dermacentor andersoni and Dermacentor variabilis, commonly occur in Canada. D. andersoni and D. variabilis are morphologically similar and are important vectors of human and animal pathogens. A practical polymerase chain reaction (PCR) assay, based on the amplification of part of the second internal transcribed spacer ribosomal DNA (pITS-2 rDNA), was developed to distinguish D. andersoni from D. variabilis. In addition, single-strand conformation polymorphism (SSCP) analysis of the pITS-2 rDNA provided a reliable method of distinguishing specimens of the three species of ixodid tick. PCR and pITS-2 SSCP were also used to determine whether there was hybridization between D. andersoni and D. variabilis at two localities in Saskatchewan where they occur in sympatry. These molecular tools will be useful for the unequivocal identification of D. andersoni and D. variabilis at all life cycle stages, which is essential for studies on their ecology and on the transmission of tick-borne diseases. Also, pITS-2 SSCP may be of potential use for discriminating among the other morphologically similar species within the genus Dermacentor.     

Stress and transcriptional regulation of tick ferritin HC

We previously identified a partial Dermacentor variabilis cDNA encoding ferritin HC (HC) subunit homolog (DVFER) that was differentially upregulated in Rickettsia montanensis infected ticks (Mulenga et al., 2003a). We have used rapid amplification of cDNA ends to clone full-length DVFER cDNA and its apparent ortholog from the wood tick, D. andersoni (DAFER), both of which show high sequence similarity to vertebrate than insect ferritin. Both DVFER and DAFER contain the stem-loop structure of a putative iron responsive element in the 5' untranslated region (nucleotide positions, 16-42) and the feroxidase centre loop typical for vertebrate ferritin HC subunits. Quantitative Western and Northern blotting analyses of protein and RNA from unfed and partially fed whole tick as well as dissected tick tissues demonstrated that DVFER is constitutively and ubiquitously expressed. Based on densitometric analysis of detected protein and mRNA bands, DVFER is predominantly expressed in the midgut, and to a lesser extent in the salivary glands, ovary and fatbody. Sham treatment (mechanical injury) and Escherichia coli challenge of D. variabilis ticks stimulated statistically significant (approximately 1.5- and approximately 3.0-fold, respectively) increases in DVFER mRNA abundance over time point matched naive control ticks. These data suggest that DVFER mRNA is nonspecifically up regulated in response to mechanical injury or bacterial infection induced stress.     

Gene organization of a novel defensin of Ixodes ricinus: first annotation of an intron/exon structure in a hard tick defensin gene and first evidence of the occurrence of two isoforms of one member of the arthropod defensin family

Antimicrobial peptides (defensins) are effectors of the immune system. Herein, we describe a novel Ixodes ricinus defensin gene(s), analyse its structure and compare it with other known antimicrobial peptides from different tick species. For the first time, an intron/exon structure is discovered in a hard-tick defensin gene. The intron/exon genomic organization of the gene is similar to the organization in Ornithodoros moubata, but not to that of the intronless defensins of Dermacentor variabilis and Ixodes scapularis. The analysis of the deduced amino acid sequences of different recombinants from the I. ricinus cDNA library reveals the presence of two defensin isoforms with three amino acid substitutions. Whether or not these substitutions affect the biological properties of the peptides is currently unknown. The expression of the defensin gene is strongly induced in the tick midgut after infection with Borrelia burgdorferi.     

Identification and characterization of a homologue of the pro-inflammatory cytokine Macrophage Migration Inhibitory Factor in the tick, Amblyomma americanum

Studying tick feeding and digestion, we discovered in a cDNA library from partially fed Amblyomma americanum ticks the first known arthropod homologue of a human cytokine, the pro-inflammatory Macrophage Migration Inhibitory Factor (MIF). The tick origin of the MIF cDNA clone was confirmed by sequencing a genomic fragment that contained the full-length tick MIF gene with two introns. Antiserum to a tick MIF-specific peptide as well as antiserum to complete tick MIF revealed the expression of tick MIF in the salivary gland and midgut tissues of A. americanum ticks. In an in vitro functional assay, recombinant tick MIF inhibited the migration of human macrophages to the same extent that recombinant human MIF did.     

Four serine proteinases expressed in Manduca sexta haemocytes

Several putative serine proteinases were detected in Manduca sexta larval plasma by labelling with radio-active diisopropyl fluorophosphate. To begin to identify and characterize such enzymes, a polymerase chain reaction was carried out using haemocyte cDNA as template and primers designed to amplify conserved sequences from serine proteinases. Four serine proteinase cDNA fragments were cloned. These were used as probes to screen an M. sexta larval haemocyte cDNA library to obtain full-length clones encoding haemocyte proteinases 1–4 (HP1, HP2, HP3 and HP4). HP1 and HP2 contain an aminoterminal ‘clip’ domain similar to those found in horse-shoe crab clotting enzyme and clotting factor B and also in the Drosophila melanogaster proteinases snake and easter. HP3 and HP4 are most similar to proteinases from mammalian leucocytes. HP1 and HP2 are both present in plasma. HP1 is expressed in haemocytes (granular cells and oenocytoids) and not in fat body. HP2 is expressed in fat body and in granular haemocytes, plasmatocytes and oenocytoids. After injection of larvae with bacteria, the level of HP2 mRNA decreased in haemocytes and increased in fat body.     

THE EXPERIMENTAL TRANSMISSION OF COLORADO TICK FEVER

1. The symptoms, history of tick bite, characteristic fever curve, and white blood cell picture should enable the physician to make a diagnosis of Colorado tick fever in nearly every case. 2. The typical white blood cell picture is a depression of the total leucocytes with a shift to the left of the granulocytes. Basophilic cytoplasmic bodies appear occasionally in lymphocytes 3 to 4 days after clinical recovery. 3. The disease can be transmitted serially in human beings by parenteral injection of blood or serum. Such transfers have not resulted in decreased or increased virulence. 4. The naturally acquired and experimental cases of Colorado tick fever are identical in their manifestations. 5. An attack of Colorado tick fever confers a degree of definite immunity to the disease. 6. Colorado tick fever is not a mild form of Rocky Mountain spotted fever since individuals immunized with ground tick vaccine against Rocky Mountain spotted fever are still susceptible to Colorado tick fever. 7. Adult Dermacentor andersoni ticks allowed to feed on typical cases, then carried through to a new generation and fed on susceptible adults, failed to transmit the disease. 8. Colorado tick fever has been successfully transmitted to an experimental animal, the golden hamster.     

Participation of haemocytes in fat body degradation via cathepsin L expression

Insect haemocytes are known to participate in innate immunity via the phagocytosis of pathogens. However, the function of haemocytes in tissue remodelling is less understood. We report here that haemocytes play roles in fat body degradation by expressing a cysteine proteinase cathepsin L in the lepidopteran Helicoverpa armigera. During metamorphosis, haemocytes undergo morphological changes by increasing their cell size and transforming their granulocytes into macrogranulocytes. The population of haemocytes also changes with increased number of granulocytes and decreased plasmatocytes. The expression level of cathepsin L in haemocytes, mainly in granulocytes and plasmatocytes, increases. The steroid hormone 20-hydroxyecdysone is able to promote the transformation of granulocytes into macrogranulocytes, and up-regulate the expression level of cathepsin L. The knock-down of the cathepsin L gene by RNA interference in haemocytes in vitro results in deficient granulocytes transforming into macrogranulocytes. Haemocytes are able to enter the decomposed fat body during metamorphosis. The over-expression of the proteinase domain C1A of cathepsin L results in cell apoptosis. Haemocytes, especially macrogranulocytes, undergo apoptosis and cathepsin L is released into haemolymph and the fat body during metamorphosis for fat body decomposition and degradation. These results suggest that cathepsin L is related to the transformation of granulocytes to macrogranulocytes to enter the fat body, and induce haemocyte apoptosis for further tissue degradation.     

山东省青岛市5种蜱携带的优势微生物种群鉴定分析

目的 分析山东省青岛市蜱携带的优势微生物种群差异，构建蜱微生物资源库。方法 收集2019年山东省青岛市野生刺猬携带的蜱，通过形态学和16S核糖体RNA测序对蜱进行分类，随后构建RNA文库，并使用Illumina NovaSeq6000进行PE2×150测序。对测序下机数据进行宏基因组分析和微生物分类学分析，并对大别班达病毒开展遗传进化分析。结果 经鉴定收集的蜱分别为褐黄血蜱（n=155）、血红扇头蜱（n=62）、中华革蜱（n=67）、长角血蜱（n=77）和铃头血蜱（n=59）。不同蜱的细菌丰度差异性大，从样本中共鉴定出6门19目35科101种细菌。褐黄血蜱和铃头血蜱中相对丰度最高的细菌为微球菌，丰度分别为37.9%和71.5%；血红扇头蜱、中华革蜱和长角血蜱中相对丰度最高的细菌均为假单胞菌，丰度分别为51.9%、49.6%和34.8%。褐黄血蜱、中华革蜱和铃头血蜱病毒种群的丰度为0.28%～2.82%，血红扇头蜱病毒种群丰度为22.98%，长角血蜱病毒种群丰度为59.31%。永嘉蜱病毒和大别山蜱病毒在5种蜱中均检出，其相对丰度分别为12.0%～51.4%和2.4%～12.2%;Nick...     

Bombyx mori cell line as a model of immune-system organs

We tested 11 Bombyx mori cell lines for induction of cecropin B gene (CecB) expression. After the immune challenge, CecB expression was induced in seven cell lines. A mixture of the cell-free supernatant from the immune-responsive cell lines and lipopolysaccharide activated a promoter of CecB in the non-immune-responsive cell line, indicating that secreted factor(s) is involved in CecB activation. The expressed sequence tags of one of the immune-responsive cell lines, NISES-BoMo-Cam1, contained genes encoding proteins similar to Relish, Cactus, clip-domain serine protease, serpin, lectin, peptidoglycan recognition protein, 6tox and gloverin, in addition to seven known B. mori immune-inducible genes. These results show that NISES-BoMo-Cam1 cells can be used as an in vitro model of the immune system organs of B. mori.     

Mutation scanning-based identification of larval and nymphal ticks (Acari: Ixodidae) from Richardson's ground squirrels (Spermophilus richardsonii)

The objective of this study was to identify the tick species parasitizing Richardson's ground squirrels (Spermophilus richardsonii) in southern Saskatchewan (Canada). Morphological examination of the adult ticks revealed the presence of three tick species, Ixodes sculptus, Ixodes kingi and Dermacentor andersoni. However, given the difficulties in identifying some of the larval and nymphal (immature) ticks using this approach, PCR-based single-strand conformation polymorphism (SSCP) and DNA sequence analyses of a portion of the mitochondrial (mt) 16S rRNA gene were used to determine their species identity. The results showed that each tick species had a unique set of SSCP profiles and DNA sequences using this mt marker. The species identity of larval and nymphal ticks was determined based on a comparison of these profiles and sequences with those of morphologically-identified adults. The detection of three tick species, which are known vectors of disease-causing agents, on the same host has important implications for understanding the ecology of vector-borne diseases, and provides an opportunity to examine fundamental questions regarding the structure and composition of the bacterial communities (i.e., both endosymbiotic and pathogenic species) in these ticks. This study shows the utility and benefits of using the present molecular method for the accurate identification of ticks at any stage of development.     

Transmission of Rickettsia slovaca and Rickettsia raoultii by male Dermacentor marginatus and Dermacentor reticulatus ticks to humans

We analyzed rickettsial DNA of ticks from tick-borne lymphadenopathy (TIBOLA) patients. Dermacentor marginatus (9/17) and Dermacentor reticulatus (8/17) transmitted rickettsiae to a similar extent. Rickettsia raoultii was detected in more ticks than Rickettsia slovaca. We observed the development of TIBOLA symptoms after the bite of males of both tick species.     

A cathepsin L-like proteinase is involved in moulting and metamorphosis in Helicoverpa armigera

Holometabolous insects undergo larval moulting and metamorphosis within their life cycle. A cDNA encoding the cathepsin L-like proteinase Ha-cathL has been cloned from Helicoverpa armigera . It has a sequence of 1826 bp and encodes a 550-residue protein with a molecular mass of 63 kDa. Northern blot analysis indicated that Ha-cathL is specifically expressed in haemocytes, with increased expression during larval moulting and metamorphosis. In vivo experimentation revealed that Ha-cathL is up-regulated by 20-hydroxyecdysone. Meanwhile, in situ hybridization and immunocytochemistry revealed that Ha-cathL mRNA is mainly expressed in granulocytes and plasmatocytes. Knock down of cathepsin L by RNA interference results in larvae death before pupation or the formation of a chimeric pupa containing a larval head and thorax, abnormal wings and the pupal abdomen. The reason for this is that the affected haemocytes cannot become granulated, and therefore cannot participate in fat body remodelling and wing development. These facts suggest that Ha-cathL is involved in larval moulting and metamorphosis by participating in the functioning of haemocytes.