Simona Sagona

Diversity, abundance and sex-specific expression of chemosensory proteins in the reproductive organs of the locust Locusta migratoria manilensis.

Abstract Chemosensory proteins (CSPs) are small soluble proteins often associated with chemosensory organs in insects but include members involved in other functions, such as pheromone delivery and development. Although the CSPs of the sensory organs have been extensively studied, little is known on their functions in other parts of the body. A first screening of the available databases has identified 70 sequences encoding CSPs in the oriental locust Locusta migratoria manilensis. Applying proteomic analysis, we have identified 17 of them abundantly expressed in the female reproductive organs, but only one (CSP91) in male organs. Bacterially expressed CSP91 binds fatty acids with a specificity for oleic and linoleic acid, as well as medium-length alcohols and esters. The same acids have been detected as the main gas chromatographic peaks in the dichloromethane extracts of reproductive organs of both sexes. The abundance and the number of CSPs in female reproductive organs indicates important roles for these proteins. We cannot exclude that different functions can be associated with each of the 17 CSPs, including delivery of semiochemicals, solubilization of hormones, direct control of development, or other unknown tasks.

Infectivity of DWV Associated to Flower Pollen: Experimental Evidence of a Horizontal Transmission Route

Deformed wing virus (DWV) is a honeybee pathogen whose presence is generally associated with infestation of the colony by the mite Varroa destructor, leading to the onset of infections responsible for the collapse of the bee colony. DWV contaminates bee products such as royal jelly, bee-bread and honey stored within the infected hive. Outside the hive, DWV has been found in pollen loads collected directly from infected as well as uninfected forager bees. It has been shown that the introduction of virus-contaminated pollen into a DWV-free hive results in the production of virus-contaminated food, whose role in the development of infected bees from virus-free eggs has been experimentally demonstrated. The aim of this study was twofold: (i) to ascertain the presence of DWV on pollen collected directly from flowers visited by honeybees and then quantify the viral load and (ii) determine whether the virus associated with pollen is infective. The results of our investigation provide evidence that DWV is present on pollen sampled directly from visited flowers and that, following injection in individuals belonging to the pollinator species Apis mellifera, it is able to establish an active infection, as indicated by the presence of replicating virus in the head of the injected bees. We also provide the first indication that the pollinator species Osmia cornuta is susceptible to DWV infection.

A proteomic investigation of soluble olfactory proteins in Anopheles gambiae

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector.

Odorant-binding proteins and olfactory coding in the solitary bee Osmia cornuta

Solitary bees are major pollinators but their chemical communication system has been poorly studied. We investigated olfactory coding in Osmia cornuta from two perspectives, chemical and biochemical. We identified (E)-geranyl acetone and 2-hexyl-1,3-dioxolane, specifically secreted by females and males, respectively. A transcriptome analysis of antennae revealed 48 ORs (olfactory receptors), six OBPs (odorant-binding proteins), five CSPs (chemosensory proteins), and a single SNMP (sensory neuron membrane protein). The numbers of ORs and OBPs are much lower than in the honeybee, in particular, C-minus OBPs are lacking in the antennae of O. cornuta. We have expressed all six OBPs of O. cornuta and studied their binding specificities. The best ligands are common terpene plant odorants and both volatiles produced by the bee and identified in this work.

A proteomic approach to identify seminal plasma proteins in roosters (Gallus gallus domesticus)

Considering the interest in avian semen processing and storage, the objective of this study was to identify the domestic fowl seminal plasma proteins using two-dimensional gel electrophoresis (2-DE) and mass spectrometry MS/MS. For three times in a 4-month period, seminal plasma was obtained from semen collected from four local male chickens (Gallus gallus domesticus) and prepared for two-dimensional polyacrylamide gel electrophoresis. A total of 83 spots were detected across all gels and analyzed by MALDI-TOF/TOF. Among these spots, 17 have been successfully identified. The most intensely stained spots were recognized as serum albumin, ovotransferrin, alpha-enolase, fatty acid binding protein, thioredoxin, trypsin inhibitor CITI-1 and gallinacin-9. From these proteins, two are characteristic of avian seminal plasma, the ovotransferrin and gallinacin-9, and one is specific of the Gallus species, the chicken trypsin inhibitor CITI-1.

Effect of 1,3-1,6 β-Glucan on Natural and Experimental Deformed Wing Virus Infection in Newly Emerged Honeybees (Apis mellifera ligustica).

The Western Honeybee is a key pollinator for natural as well as agricultural ecosystems. In the last decade massive honeybee colony losses have been observed worldwide, the result of a complex syndrome triggered by multiple stress factors, with the RNA virus Deformed Wing Virus (DWV) and the mite Varroa destructor playing crucial roles. The mite supports replication of DWV to high titers, which exert an immunosuppressive action and correlate with the onset of the disease. The aim of this study was to investigate the effect of 1,3–1,6 β-glucan, a natural innate immune system modulator, on honeybee response to low-titer natural and high-titer experimental DWV infection. As the effects exerted by ß-glucans can be remarkably different, depending on the target organism and the dose administered, two parallel experiments were performed, where 1,3–1,6 ß-glucan at a concentration of 0.5% and 2% respectively, was added to the diet of three cohorts of newly emerged honeybees, which were sampled from a Varroa-free apiary and harboured a low endogenous DWV viral titer. Each cohort was subjected to one of the following experimental treatments: no injection, injection of a high-copy number DWV suspension into the haemocel (experimental DWV infection) or injection of PBS into the haemocoel (physical injury). Control bees fed a ß-glucan-free diet were subjected to the same treatments. Viral load, survival rate, haemocyte populations and phenoloxidase activity of each experimental group were measured and compared. The results indicated that oral administration of 0.5% ß-glucan to naturally infected honeybees was associated with a significantly decrease of the number of infected bees and viral load they carried, and with a significant increase of the survival rate, suggesting that this natural immune modulator molecule might contribute to increase honeybee resistance to viral infection.

Palynological origin, chemical composition, lipid peroxidation and fatty acid profile of organic Tuscanian bee-pollen

The aim of this work is to characterize fresh bee-pollen in order to define the main nutritional properties of this apiary product for future legislation. Organic bee-pollen (n = 5) harvested from Lucca (Tuscany, Italy) was studied for palynological analysis, nutritional content (dry matter, crude protein, lipid, ash and carbohydrate contents), thiobarbituric acid reactive substances (TBARS) content and fatty acids (FA) profile. The chemical composition analyses showed an average of 849 g/kg for dry matter, 253 g/kg for proteins, 12.9 g/kg for lipids, 24.7 g/kg for ash, and 561 g/kg for carbohydrates. The average value of TBARS content was 0.96 mg malondialdehyde/kg. Concerning FA composition, results showed differences among organic Tuscanian bee-pollen species. All the investigated pollen species had linoleic acid with a mean value of 55.4 g/kg of total FAs and, α-linolenic acid, with a mean value of 147 g/kg of total FAs. An average n-6/n-3 polyunsaturated fatty acid (n-6/n-3 PUFA) ratio of 1.70 was found. Considering that the n-6/n-3 PUFA ratio and linoleic acid/α-linolenic acid ratio are considered important for the prevention of human diseases, the Tuscanian organic bee-pollen may be of nutraceutical relevance.

Parasitization of a wild and reared population of the solitary bee Osmia cornuta Latr. by the parasitoid Anthrax anthrax Schrank (Diptera, Bombylidae): comparison between two types of artificial nest

A nest-trapping (NT) and a releasing and rearing (RR) campaign were performed in two similar areas of Tuscany (Italy), towards nest inducing and reproduction of Osmia cornuta Latr. For the NT campaign, artificial nests built with reed segments (Arundo donax L.) (reed nests) and grooved hardboard plates were used. The latter type was termed Artificial Assembled Trap Nest (AATN). The RR campaign utilized the same nest types and a reared O. cornuta population, with 100 cocoons having a sex-ratio of 2:1 (female:male). Nests were removed from fields during the first week of August, placed in mesh bags and kept in the laboratory at room temperature up to the month of October, when each nest was opened in order to analyse its content. Results showed that in both investigated areas, the wild and reared population were parasitized by the bombylid dipteran Anthrax anthrax Schrank. A. anthrax parasitization of O. cornuta was found to be greater in assembled (AATN) compared to reed nests. Highest prevalence of parasitization was found in the innermost portion of the tunnel, where Osmia cocoons usually contains females. Additional damage is cocoon crashing, caused by A. anthrax armed pupae as it forces its way out of the tunnel. Both O. cornuta population (reared and wild) resulted parasitized by A. anthrax specimens. In October, when broodnests were opened, exuviae of newly emerged specimens, armed pupae and mature diapausing A. anthrax Schrank larvae were, in some cases, found inside together, suggesting that the life cycle of this dipteran is parsivoltine.

Complete Genome Sequence of Deformed Wing Virus Isolated from Vespa crabro in Italy

ABSTRACT In this article, we document the first isolation of a replication-competent deformed wing virus from Vespa crabro in Italy. Although the virus has never been isolated from this insect, the sequence of this virus shows a strong sequence homology with isolates obtained from Apis mellifera, which is considered its natural host.

A Serological Survey on Swine Brucellosis Using Standard Procedures, Dot Blot, and Western Blot in Finisher Pigs in Central-North Italy

In recent years, Brucella suis has been sporadically reported in Italy in domestic and wild swine. Since standard serological tests can determine false positive results, the development of alternative tests with improved sensitivity and specificity is rather essential. We analyzed 1212 sera collected at slaughterhouse from healthy pigs belonging to 62 farms of North-Central Italy. Sera were tested by Rose Bengal Test, Complement Fixation Test, and subsequently by a Dot Blot (DB) and Western Blot assays (WB). Only one serum resulted positive to all tests, indicating that swine brucellosis has a very limited spread. DB and WB could represent a support to the available serological tests; however, further studies to validate these tests are needed. In the presence of reemerging diseases, a prompt and continuous monitoring design is necessary to acquire epidemiological information for the subsequent application of specific health emergency plans.