Release of Mediator Enzyme ß-Hexosaminidase and Modulated Gene Expression Accompany Hemocyte Degranulation in Response to Parasitism in the Silkworm Bombyx mori.

In insects infections trigger hemocyte-mediated immune reactions including degranulation by exocytosis; however, involvement of mediator enzymes in degranulation process is unknown in insects. We report here that in silkworm Bombyx mori, infection by endoparasitoid Exorista bombycis and microsporidian Nosema bombycis activated granulation in granulocytes and promoted degranulation of accumulated structured granules. During degranulation the mediator lysosomal enzyme ß-hexosaminidase showed increased activity and expression of ß-hexosaminidase gene was enhanced. The events were confirmed in vitro after incubation of uninfected hemocytes with E. bombycis larval tissue protein. On infection, cytotoxicity marker enzyme lactate dehydrogenase (LDH) was released from the hemocytes illustrating cell toxicity. Strong positive correlation (R2 = 0.71) between LDH activity and ß-hexosaminidase released after the infection showed parasitic-protein-induced hemocyte damage and accompanied release of the enzymes. Expression of ß-hexosaminidase gene was enhanced in early stages after infection followed by down regulation. The expression showed positive correlation (R2 = 0.705) with hexosaminidase activity pattern. B. mori hexosaminidase showed 98% amino acid similarity with that of B. mandarina showing origin from same ancestral gene; however, 45-60% varied from other lepidopterans showing diversity. The observation signifies the less known association of hexosaminidase in degranulation of hemocytes induced by parasitic infection in B. mori and its divergence in different species.

Comparative Analysis of Coding and Non-Coding Features within Insect Tolerance Loci in Wheat with Their Homologs in Cereal Genomes.

Food insecurity and malnutrition have reached critical levels with increased human population, climate fluctuations, water shortage; therefore, higher-yielding crops are in the spotlight of numerous studies. Abiotic factors affect the yield of staple food crops; among all, wheat stem sawfly (Cephus cinctus Norton) and orange wheat blossom midge (Sitodiplosis mosellana) are two of the most economically and agronomically harmful insect pests which cause yield loss in cereals, especially in wheat in North America. There is no effective strategy for suppressing this pest damage yet, and only the plants with intrinsic tolerance mechanisms such as solid stem phenotypes for WSS and antixenosis and/or antibiosis mechanisms for OWBM can limit damage. A major QTL and a causal gene for WSS resistance were previously identified in wheat, and 3 major QTLs and a causal gene for OWBM resistance. Here, we present a comparative analysis of coding and non-coding features of these loci of wheat across important cereal crops, barley, rye, oat, and rice. This research paves the way for our cloning and editing of additional WSS and OWBM tolerance gene(s), proteins, and metabolites.

Improved transgenic sexing strains for genetic control of the Australian sheep blow fly Lucilia cuprina using embryo-specific gene promoters.

For genetic approaches for controlling insect pests such as the sterile insect technique (SIT), it is advantageous to release only males as females are ineffective as control agents and they consume about 50% of the diet. Here we developed tetracycline-repressible Lucilia cuprina transgenic strains in which adult females were fully fertile and viable on a diet that lacked tetracycline and all of their female offspring died at the embryo stage. The transgenic strains are an improvement over the strains we developed previously, which had the disadvantage that adult females on diet without tetracycline were sterile and died prematurely. This was possibly due to the low level expression of the effector gene in ovaries. In the strains developed in this study, the early promoters from L. cuprina nullo or Cochliomyia macellaria CG14427 genes were used to drive the tetracycline transactivator (tTA) expression in the early embryo. In the absence of tetracycline, tTA activates expression of the proapoptotic gene Lshid which contains a female-specific intron. Consequently, only females produce active HID protein and die at the embryo stage. Crossing the tTA-expressing driver lines with an RFPex reporter line confirmed that there was no expression of the effector gene in the ovary. These new embryonic L. cuprina transgenic sexing strains hold great promise for genetic control programs and the system reported here might also be transferable to other major calliphorid livestock pests such as the New World screwworm, Cochliomyia hominivorax.

High morbidity cutaneous enzootic myiasis by Dermatobia hominis (Diptera: Oestridae) in sambar deer (Rusa unicolor).

This study describes the first known occurrence and treatment of enzootic cutaneous myiasis by Dermatobia hominis in a herd of sambar deer (Rusa unicolor). Sambar deer are clinically affected by enzootic cutaneous myiasis during the spring and summer in Rio de Janeiro, Brazil. Through direct inspection and clinicopathological evaluation in the herd with 80 R. unicolor, it was observed that infestation by D. hominis caused 100% morbidity. At histological exam, skin had eosinophilic and granulomatous chronic active severe necrohemorrhagic dermatitis associated with botfly and moderate surrounding fibroplasia. The systemic treatment with oral ivermectin at 0.08% was effective in 93.7% of deer with enzootic cutaneous myiasis after fourteen days. After treatment, the skin deer had eosinophilic and granulomatous chronic severe dermatitis associated with degenerated botfly and severe fibroplasia. Notable differences in leukocyte profile were observed between groups pre- and post-treatment. Decrease of relative values of neutrophils and eosinophils were significant in the treated deer group. An increase of relative values of monocytes was also confirmed in the treated group. Sambar deer was the only species affected by D. hominis, even though several other wild herbivores were kept in the same area. More studies are needed to elucidate the susceptibility of R. unicolor to D. hominis cutaneous infestations.

[Myases from here and elsewhere†: pseudo-furonculosis and Ignatzschineria larvae bacteremia]. / Myiases d'ici et d'ailleurs†: pseudo-furonculose et bactériémie à Ignatzschineria larvae.

Myiasis is an infestation by maggots. In humans, it predominates in regions with low socio-economic development. We report on two cases of myiasis acquired during a tropical travel and in Switzerland, respectively. The first one presented as a furunculous-like disease due to the invasion of subcutaneous tissues by Cordylobia sp. larvae. The second corresponded to a chronic wound infestation that resulted in a rarely reported bacteremia due to Ignatzschineria larvae, a commensal bacteria of maggots' digestive tract. Surgery was necessary in both cases, mainly for psychological reasons in the first case. Both the entomologist and molecular biology were instrumental for treatment decisions.

La myiase est une infestation par des larves de mouches. Chez l'homme, elle prédomine dans les régions à faible niveau socio-économique. Nous rapportons ici deux cas de myiase, l'un acquis lors d'un voyage sous les tropiques et l'autre autochtone : une myiase furonculaire due à la pénétration d'une larve de diptère dans la peau, en l'occurrence Cordylobia sp. ; et une myiase de plaie survenue par ponte de mouches dans des tissus nécrotiques, avec une exceptionnelle bactériémie secondaire, due à une bactérie commensale du tractus digestif de ces larves, Ignatzschineria larvae. Dans les deux situations, la chirurgie a été nécessaire, pour une indication surtout d'ordre psychologique dans la première. Dans les deux cas, l'apport de l'entomologiste et de la biologie moléculaire a été déterminant dans la décision thérapeutique.

A Case of Cutaneous Myiasis Caused by Cordylobia anthropophaga Larvae in a Korean Traveler Returning from Central Africa.

The cutaneous myiasis has been rarely reported in the Republic of Korea. We intended to describe here a case of furuncular cutaneous myiasis caused by Cordylobia anthropophaga larvae in a Korean traveler returned from Central Africa. A patient, 55-year-old man, had traveled to Equatorial Guinea, in Central Africa for a month and just returned to Korea. Physical examinations showed 2 tender erythematous nodules with small central ulceration on the left buttock and thigh. During skin biopsy, 2 larvae came out from the lesion. C. anthropophaga was identified by paired mouth hooks (toothed, spade-like, oral hooklets) and 2 posterior spiracles, which lack a distinct chitinous rim. Although rarely described in Korea until now, cutaneous myiasis may be encountered more frequently with increasing international travel and exchange workers to tropical areas.

First record of soybean stem fly Melanagromyza sojae (Diptera: Agromyzidae) in Paraguay confirmed by molecular evidence.

We provided the first scientific record of Melanagromyza sojae (Zehntner, 1900), through molecular characterization of partial mtDNA COI gene, that confirms the occurrence of this pest in Paraguay. Previously reported in Brazil, an outbreak of larvae of M. sojae known as the soybean stem fly (SSF) that belongs to the family Agromyzidae, was also noted in soybean fields from the Canindeyú, Alto Paraná and Itapúa Departments in Paraguay. This pest is highly polyphagous, attacking various host plant species from the family Fabaceae, such as soybean and other beans. The implications of SSF detection in Paraguay are discussed in relation to the current soybean cultivation practices from this agriculturally important South American region, including Brazil.

Analysis of SSH library of rice variety Aganni reveals candidate gall midge resistance genes.

The Asian rice gall midge, Orseolia oryzae, is a serious insect pest causing extensive yield loss. Interaction between the gall midge and rice genotypes is known to be on a gene-for-gene basis. Here, we report molecular basis of HR- (hypersensitive reaction-negative) type of resistance in Aganni (an indica rice variety possessing gall midge resistance gene Gm8) through the construction and analysis of a suppressive subtraction hybridization (SSH) cDNA library. In all, 2,800 positive clones were sequenced and analyzed. The high-quality ESTs were assembled into 448 non-redundant gene sequences. Homology search with the NCBI databases, using BlastX and BlastN, revealed that 73% of the clones showed homology to genes with known function and majority of ESTs belonged to the gene ontology category 'biological process'. Validation of 27 putative candidate gall midge resistance genes through real-time PCR, following gall midge infestation, in contrasting parents and their derived pre-NILs (near isogenic lines) revealed induction of specific genes related to defense and metabolism. Interestingly, four genes, belonging to families of leucine-rich repeat (LRR), heat shock protein (HSP), pathogenesis related protein (PR), and NAC domain-containing protein, implicated in conferring HR+ type of resistance, were found to be up-regulated in Aganni. Two of the reactive oxygen intermediates (ROI)-scavenging-enzyme-coding genes Cytosolic Ascorbate Peroxidase1, 2 (OsAPx1 and OsAPx2) were found up-regulated in Aganni in incompatible interaction possibly suppressing HR. We suggest that Aganni has a deviant form of inducible, salicylic acid (SA)-mediated resistance but without HR.

Genes Expressed Differentially in Hessian Fly Larvae Feeding in Resistant and Susceptible Plants.

The Hessian fly, Mayetiola destructor, is a destructive pest of wheat worldwide and mainly controlled by deploying resistant cultivars. In this study, we investigated the genes that were expressed differentially between larvae in resistant plants and those in susceptible plants through RNA sequencing on the Illumina platform. Informative genes were 11,832, 14,861, 15,708, and 15,071 for the comparisons between larvae in resistant versus susceptible plants for 0.5, 1, 3, and 5 days, respectively, after larvae had reached the feeding site. The transcript abundance corresponding to 5401, 6902, 8457, and 5202 of the informative genes exhibited significant differences (p ≤ 0.05) in the respective paired comparisons. Overall, genes involved in nutrient metabolism, RNA and protein synthesis exhibited lower transcript abundance in larvae from resistant plants, indicating that resistant plants inhibited nutrient metabolism and protein production in larvae. Interestingly, the numbers of cytochrome P450 genes with higher transcript abundance in larvae from resistant plants were comparable to, or higher than those with lower transcript abundance, indicating that toxic chemicals from resistant plants may have played important roles in Hessian fly larval death. Our study also identified several families of genes encoding secreted salivary gland proteins (SSGPs) that were expressed at early stage of 1(st) instar larvae and with more genes with higher transcript abundance in larvae from resistant plants. Those SSGPs are candidate effectors with important roles in plant manipulation.

New insight into the Delia platura Meigen caused alteration in nutrient content of soybean (Glycine max L. Merill).

Climate change has brought about an increasing level of seedcorn maggot (Delia platura Meigen, 1826) (Diptera: Anthomyiidae) damage in Hungary. In order to have a more accurate understanding of the effects of these plant injuries induced by the larvae of D. platura, the nutrient content of soybean (Glycine max L. Merill.) was studied. Our results show that the moisture, raw fat, raw fibre, and raw ash content of the batches damaged by D. platura were significantly less in comparison with that of the control samples. In response to the deleterious effect of the insect, the infected soybean plants showed forced ripening (P = 0.004) (P > 0.05). The difference of moisture content between damaged and control samples was 2.30% on average. The fact of nutritional value loss was also reflected by the alteration of sugar mobilisation. As the result of this experiment the sucrose breakdown to glucose and fructose during the germination was significantly slower in the damaged seeds than that of the control ones. Overall, this late and surprising damage caused by D. platura led to the forced ripening of the affected soybean plants and a significant change in their nutritional values. Based on the herein reported results, it is presumable that in cases when the current climatic extremities, which are envisaged to occur more frequently in the future, and effects of agricultural practices will be coincided in the future a qualitative change of the produced soybean batches can be expected through the damage caused by this fly species.

[Comparison of the temperature-driven seasonality of campylobacteriosis and salmonellosis and the annual phenology of Eristalis tenax (Diptera: Syrphidae)]. / A campylobacteriosis és a salmonellosis heti átlaghomérséklet által meghatározott szezonalitásának és az Eristalis tenax (Diptera: Syrphidae) éves fenológiájának összehasonlítása.

INTRODUCTION: Ambient temperature and the activity of Diptera species are the primary factors of the seasonality of bacterial enteral diseases. AIM: The authors analyzed the effect of the weekly mean ambient temperature on salmonellosis and campylobacteriosis incidence and the annual phenology of the potential vector Eristalis tenax. METHOD: Weekly case number data of the period between 2004 and 2014 were derived from the Hungarian National Center for Epidemiology. European Climate Assessment Dataset was the source of the weekly mean temperature data for the grid overlapping Hungary. RESULTS: While in the case of campylobacteriosis weak correlation was found (r(2) = 0.39), salmonellosis showed strong correlation with mean temperature (r(2) = 0.71) using 8-weeks lag before the outbreak of the cases. CONCLUSIONS: Comparing the mean weekly incidence of campylobacteriosis and salmonellosis with the modeled weekly activity of Eristalis tenax it was found that vector Diptera species may influence the incidence of enteric diseases in late spring and summer, in July and August particularly.

[Fauna of Rare and Poorly Studied Ectoparasites of bats in Southern Siberia].

Forty-six chiropteran specimens from seven species have been captured on the territory of Southern Siberia. From them, 676 ectoparasites of 16 species (gamasid mites and insects) were collected. The bat fly Basilia mongolensis mongolensis Theodor, 1966 was found in Russia for the first time. The gamasid mites Spinturnix maedai Uchikawa et Wada, 1979 and Macronyssus hosonoi Uchikawa, 1979 have not previously been registered in Siberia. Several species have been recorded on new hosts. Three species of gamasid mites out of the genus Macronyssus are, probably, new to science. New data on the ecology of low-abundant and understudied bat species belonging to the Siberian-Far Eastern complex have been provided.

Virulence and biotype analyses of Hessian fly (Diptera: Cecidomyiidae) populations from Texas, Louisiana, and Oklahoma.

Hessian fly, Mayetiola destructor (Say, 1817), is a major pest of wheat, and is controlled mainly through deploying fly-resistant wheat cultivars. The challenge for the plant resistance approach is that virulence of Hessian fly populations in the field is dynamic, and wheat cultivars may lose resistance within 6-8 yr. To ensure continuous success of host plant resistance, Hessian fly populations in the field need to be constantly monitored to determine which resistance genes remain effective in different geographic regions. This study investigated five Hessian fly populations collected from Texas, Louisiana, and Oklahoma, where infestation by Hessian fly has been high in recent years. Eight resistance genes, H12, H13, H17, H18, H22, H25, H26, and Hdic, were found to be highly effective against all tested Hessian fly populations in this region, conferring resistance to > or = 80% of plants containing one of these resistance genes. The frequencies ofbiotypes virulent to resistance genes H13 (biotype vH13), H18 (vH18), H21 (vH21), H25 (vH25), H26 (vH26), and Hdic (vHdic) were determined, and were found to vary from population to population, ranging from 0 to 45%. A logistic regression model was established to predict biotype frequencies based on the correlation between the percentages of susceptible plants obtained in a virulence test and the log-odds of virulent biotype frequencies determined by a traditional approach.

Myiasis.

Myiasis is defined as the infestation of live vertebrates (humans and/or animals) with dipterous larvae. In mammals (including humans), dipterous larvae can feed on the host's living or dead tissue, liquid body substance, or ingested food and cause a broad range of infestations depending on the body location and the relationship of the larvae with the host. In this review, we deeply discuss myiasis as a worldwide infestation with different agents and with its broad scenario of clinical manifestations as well as diagnosis techniques and treatment.

[Creation of transgenic sugar beet lines expressing insect pest resistance genes cry1C and cry2A].

Impact of insect pests makes a significant limitation of the sugar beet crop yield. Integration of cry-genes of Bacillus thuringiensis into plant genome is one of the promising strategies to ensure plant resistance. The aim of this work was to obtain sugar beet lines (based on the MM 1/2 line) transformed with cry2A and cry1Cgenes. We have optimized transformation protocol and direct plant let regeneration protocol from leaf explants using 1 mg/l benzylaminopurine as well as 0,25 mg/l benzylaminopurine and 0,1 mg/l indole-butyric acid. Consequently, transgenic sugar beet lines transformed with vector constructs pRD400-cry1C and pRD400-cry2A have been obtained. PCR analysis revealed integration of cry2A and cry1C into genome of transgenic lines and expression of these genes in leaf tissues was shown by reverse transcription PCR.

Infancy Urogenital Myiasis: A Case Report.

Myiasis is a parasitic infestation of fly larvae caused by dipterous larvae that live in the host's dead or living tissues. This study reported the first case of urogenital myiasis in infants from Iran caused by Lucilia sericata. The 10-month-old boy was taken to the hospital by his mother due to restlessness and abdominal pain. During the examination, motile and live larvae were observed in the penis area. The patient had no history of infectious disease or immune system deficiency, and his blood tests were normal. The entomological analysis found that the source of this myiasis was Lucilia sericata fly larvae. The report of such cases highlighted the importance of physical examination and the awareness of physicians in dealing with myiasis, as well as the necessity of educating mothers on the hygiene of the living environment and child care in rural areas.

Deep sequencing and genome-wide analysis reveals the expansion of MicroRNA genes in the gall midge Mayetiola destructor.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in regulating post transcriptional gene expression. Gall midges encompass a large group of insects that are of economic importance and also possess fascinating biological traits. The gall midge Mayetiola destructor, commonly known as the Hessian fly, is a destructive pest of wheat and model organism for studying gall midge biology and insect - host plant interactions. RESULTS: In this study, we systematically analyzed miRNAs from the Hessian fly. Deep-sequencing a Hessian fly larval transcriptome led to the identification of 89 miRNA species that are either identical or very similar to known miRNAs from other insects, and 184 novel miRNAs that have not been reported from other species. A genome-wide search through a draft Hessian fly genome sequence identified a total of 611 putative miRNA-encoding genes based on sequence similarity and the existence of a stem-loop structure for miRNA precursors. Analysis of the 611 putative genes revealed a striking feature: the dramatic expansion of several miRNA gene families. The largest family contained 91 genes that encoded 20 different miRNAs. Microarray analyses revealed the expression of miRNA genes was strictly regulated during Hessian fly larval development and abundance of many miRNA genes were affected by host genotypes. CONCLUSION: The identification of a large number of miRNAs for the first time from a gall midge provides a foundation for further studies of miRNA functions in gall midge biology and behavior. The dramatic expansion of identical or similar miRNAs provides a unique system to study functional relations among miRNA iso-genes as well as changes in sequence specificity due to small changes in miRNAs and in their mRNA targets. These results may also facilitate the identification of miRNA genes for potential pest control through transgenic approaches.

Cephenemyia stimulator and Hypoderma diana infection of roe deer in the Czech Republic over an 8-year period.

A survey of naso-pharyngeal and subcutaneous myiasis affecting roe deer (Capreolus capreolus) was conducted in the Czech Republic over an 8-year period (1999-2006). A total of 503 bucks and 264 does from six hunting localities were examined. The sampling area comprised predominantly agricultural lowlands and a mountain range primarily covered by forest. Since 1997, the deer have been treated each winter across the board with ivermectin (150 mg/kg, CERMIX® pulvis, Biopharm, CZ). Parasites found were the larvae of Hypoderma diana and Cephenemyia stimulator. There were no significant differences in warble fly infection among captured animals in the individual hunting localities. Overall, 146 (28.8%) of 503 animals (bucks) were infected with Cephenemyia stimulator larvae; body size of the second instar larva reached 13-18 mm. The prevalence ranged from 16.1 to 42.9% per year, and the mean intensity from 6 to 11 larvae per animal. Additionally, a total of 264 roe deer (does) were examined for H. diana larvae, and 77 (29.1%) were found to be positive; body size of the second instar larva reached 17 mm. The prevalence ranged from 18.8 to 50.0% per year, and the mean intensity from 13 to 22 larvae per animal. The results showed that the bot flies, Cephenemyia stimulator as well as H. diana, are common parasites in roe deer in the Czech Republic, and that through the help of treatment (ivermectin), it is possible to keep parasite levels low. The body weights of infected and non-infected H. diana deer did not differ significantly.

New species of Tetradiplosis (Diptera: Cecidomyiidae) inducing galls on Prosopis caldenia (Fabaceae) in Argentina.

Two new species of Tetradiplosis inducing galls on Prosopis caldenia are described from Argentina: Tetradiplosis panghitruz Martínez n. sp. and Tetradiplosis rayen Martínez n. sp. Tetradiplosispanghitruz induces multilocular galls on vegetative stems, whereas T. rayen induces unilocular galls containing multiple larvae on the rachis of the developing inflorescences. The adult male, female, pupa and larva are described and illustrated for both species. A key to the known species of the genus is provided.