Francesco Porcelli

Infectivity and Transmission of Xylella fastidiosa by Philaenus spumarius (Hemiptera: Aphrophoridae) in Apulia, Italy

ABSTRACT Discovery of Xylella fastidiosa from olive trees with “Olive quick decline syndrome” in October 2013 on the west coast of the Salento Peninsula prompted an immediate search for insect vectors of the bacterium. The dominant xylem-fluid feeding hemipteran collected in olive orchards during a 3-mo survey was the meadow spittlebug, Philaenus spumarius (L.) (Hemiptera: Aphrophoridae). Adult P. spumarius, collected in November 2013 from ground vegetation in X. fastidiosa-infected olive orchards, were 67% (40 out of 60) positive for X. fastidiosa by polymerase chain reaction (PCR) assays. Euscelis lineolatus Brullé were also collected but tested negative for the pathogen. Transmission tests with P. spumarius collected from the Salento area were, therefore, conducted. After a 96-h inoculation access period with 8 to 10 insects per plant and a 30-d incubation period, PCR results showed P. spumarius transmitted X. fastidiosa to two of five periwinkle plants but not to the seven olive plants. Sequences of PCR products from infected periwinkle were identical with those from X. fastidiosa-infected field trees. These data showed P. spumarius as a vector of X. fastidiosa strain infecting olives trees in the Salento Peninsula, Italy.

ISOLATION OF A XYLELLA FASTIDIOSA STRAIN INFECTING OLIVE AND OLEANDER IN APULIA, ITALY

SUMMARY The isolation in pure culture of the Xylella fastidiosa strain associated with the quick decline syndrome of olive, recently observed in Apulia (Salento peninsula, southern Italy) was attempted from symptomatic, naturally infected olive and oleander plants, and a periwinkle seedling that had been exposed to, and was infected by Xylella-positive spittlebugs. Prior to isolation, the presence of Xylella was ascertained in all donor hosts by PCR, indirect immunofluorescence and electron microscopy. Isolations from olive failed because of the heavy contamination by bacteria other than Xylella. By contrast, pure bacterial cultures were obtained from oleander and periwinkle extracts plated in periwinkle wilt gelrite (PWG) and buffered cysteine-yeast extract (BCYE) media. In both media, colonies were slow-growing, small-sized (less than 1 mm 25 days from plating), non pigmented, opalescent and exhibited the same morphology, except for the margin that was entire in BCYE and somewhat irregular in PWG. Bacterial cells were rod-shaped with rounded ends, had a thick and rippled cell wall, an average width of 0.35 µm, and a maximum length of ca. 5 µm. They gave a positive reaction in immunofluorence assays and were clearly decorated by colloidal gold in immunogold labelling tests. Sequenced PCR products amplified from periwinkle and oleander colonies shared 97-99% sequence identity with known X. fastidiosa strains from database and were 100% identical to one another and to comparable sequences obtained from infected olive trees. These sequences grouped in a distinct cluster of a branch comprising X. fastidiosa isolates belonging to the subspecies pauca.

The bodies of two missing children in an enclosed underground environment

Two young children, respectively 11 and 13 years old, originally from a small town of Southern Italy, were missing on 5 June 2006 (06:30 p.m.). Soon after their disappearance, the search for the missing children began. Broadcasters promptly transmitted a description of the missing children, pushing the entire community to assist in the search and safe recovery of the children. However, every effort was in vain, and the search went on for more than 1 year. During the missing-child search, the investigators collected enough evidence against the father, who was arrested 17 months after the childrens disappearance. He was indicted for kidnapping, homicide and concealment of the two bodies. He never confessed to the crimes and claimed to be innocent. Three months after the conviction, a fireman found the two corpses in a subterranean, dry cistern next to a well over 20 m deep. The bodies were well preserved, almost mummified, with only few body-parts skeletonised. Based on dental records, they were identified as those of the two children, who had gone missing 1.5 years before. Signs of a very low insect activity were present, reasonably consistent with a rapid skin dehydration. The autopsy showed no signs of defence injuries or ligature consistent with strangulation or captivation, except for fractures of the axial skeleton at a number of points consistent with a fall from a low-medium height. The body of the elder brother presented major injuries with signs of recent haemorrhages and gut content analysis consistent with the last meal, which provided enough evidence to ascertain a very short survival time. The younger child showed minor injuries, signs of old haemorrhages and gastric- and intestine-emptying time consistent with a longer survival time of approximately 3-4 days, spent alone in the dark and cold cistern. A long post-mortem interval (PMI) of approximately 20 months was estimated, mainly from the pattern of insect succession. Based on such physical evidence, on 4 March 2008, 4 months after conviction and 40 days after the recovery of the two bodies, the father was finally released from prison and exonerated from previous indictment of homicide.

Successional patterns of the insect fauna on a pig carcass in southern Italy and the role of Crematogaster scutellaris (Hymenoptera, Formicidae) as a carrion invader

The sarcosaprophagous fauna plays a key role in organic matter decomposition. Moreover, the biological, ecological and behavioral specificities of the taxa are useful to reconstruct the decay history of a corpse or carcass, often back to the lethal event. Here we report the seasonal succession of the insect fauna on a pig carcass exposed in a rural area in Calabria (southern Italy) during summer 2007 and 2008. The aim is to identify and qualitatively assess the major taxa of forensic importance in this region. The principal fly invaders were Lucilia caesar (L.), L. sericata (Meigen, 1826), Chrysomya albiceps (Wiedemann, 1819), Sarcophaga (Meigen, 1826) spp. and Amobia (Robineau‐Desvoidy, 1830) spp., Musca domestica (L.) and Muscina stabulans (Fallen, 1817). The primary beetle species collected in summer belonged to Dermestidae, Dermestes maculatus (De Geer, 1774) and Cleridae, Necrobia rufipes (De Geer, 1775). This paper also examined the ecological role of ants in the insect succession and describes the evidence of skin injuries directly inflicted by the acrobat ant Crematogaster (Acrocoelia) scutellaris (Olivier, 1791) (Hymenoptera Formicidae) while feeding on pig carrion. Ants belonging to two other species were also collected: Camponotus aethiops (Latreille, 1798) and Tetramorium semilaeve (André, 1881). Ants can invade carcasses and corpses directly, disrupting blowfly egg laying or preying on their larvae. Our data on the carrion faunal composition and role of ants as invaders should be useful for further forensic cases in Calabria (southern Italy). This is among the few reports of ants as forensically relevant species.

Spittlebugs as vectors of Xylella fastidiosa in olive orchards in Italy

The recent introduction of Xylella fastidiosa in Europe and its involvement in the Olive Quick Decline Syndrome (OQDS) in Apulia (Salento, Lecce district, South Italy) led us to investigate the biology and transmission ability of the meadow spittlebug, Philaenus spumarius, which was recently demonstrated to transmit X. fastidiosa to periwinkle plants. Four xylem-sap-feeding insect species were found within and bordering olive orchards across Salento during a survey carried out from October 2013 to December 2014: P. spumarius was the most abundant species on non-olive vegetation in olive orchards as well as on olive foliage and was the only species that consistently tested positive for the presence of X. fastidiosa using real-time PCR. P. spumarius, whose nymphs develop within spittle on weeds during the spring, are likely to move from weeds beneath olive trees to olive canopy during the dry period (May to October 2014). The first X. fastidiosa-infective P. spumarius were collected in May from olive canopy: all the individuals previously collected on weeds tested negative for the bacterium. Experiments demonstrated that P. spumarius transmitted X. fastidiosa from infected to uninfected olive plants. Moreover, P. spumarius acquired X. fastidiosa from several host plant species in the field, with the highest acquisition rate from olive, polygala and acacia. Scanning electron microscopy (SEM) revealed bacterial cells resembling X. fastidiosa in the foreguts of adult P. spumarius. The data presented here are essential to plan an effective IPM strategy and limit further spread of the fastidious bacterium.

Blowing a nose black and blue

In June, 2006, a 32-year-old woman, working as an architect at the archaeological site of Pompeii, noticed that her nose was blocked, especially the right nostril. When she blew her nose, the mucus was black. A few days later, she developed a dry cough and intermittent fever. Her general practitioner referred her to an otolaryngologist. Nasal examination showed no abnormality, other than mild septal deviation. Skin-prick tests for allergy gave negative results, as did nasal smears for bacteria and fungi; CT of the sinuses showed nothing of note. Nonetheless, the patient’s nasal blockage and rhinorrhoea worsened; she had occasional fever spikes, and was referred to an infectious-diseases specialist. Chest radiography and pulmonary function tests gave normal results; blood tests showed a high concentration of C-reactive protein, mild neutrophilia, and, although no fungal hyphae had been found in nasal smears, antibodies to aspergillus. Oral itraconazole (100 mg daily) was therefore prescribed; 2 months later, no benefi t had been observed. We saw the patient in September, 2007. Nasal endoscopy, CT of the sinuses, and allergy tests again gave negative results. Nasal smears showed no fungi, but we observed black, amorphous bodies, which we interpreted as cellular debris (fi gure). We suggested that the patient clean her nose and sinuses with a douche, which allowed a continuous fl ow of saline through the nose. During the fi rst washing, a small insect was expelled from the nostril; immediate symptomatic improvement ensued. The patient informed us, and the insect was collected and identifi ed as Clogmia albipunctata [=Telmatoscopus albipunctatus]. We deduced that the amorphous black objects were insect faeces. The fever, cough, and nasal obstruction completely resolved within a few days—however, the black discharge continued, although, given the mucociliary clearance rate, we had expected it to disappear in about 2 days. The patient became depressed and frustrated, and feared that she had other insects in her nose, although this possibility was excluded by CT and MRI. During a consultation, we noticed that she wore eyeliner. She confi rmed that she sometimes used a kohl pencil. We asked her to stop. The black secretion disappeared within 2 days. We realised that kohl, mixed with tears, had been passing though an abnormally patent nasolacrimal duct, and produced the blackpigmented bodies previously identifi ed as insect faeces. When the patient wore blue eyeliner, blue nasal secretions appeared. Our lessons from this event? When usual diagnoses for common illnesses such as rhinitis are excluded, refl ect on underlying mechanisms, and consider the (nearly) impossible. In this case, the nasolacrimal duct was, unusually, wide enough to host an insect, and to allow kohl fragments to enter the nose. What caused the systemic infl ammation? The patient did not have conjunctivitis, although the conjunctiva was in direct contact with her eyeliner. Moreover, the infl ammatory symptoms stopped after the fl y was ejected. We therefore blame the fl y. We note, in passing, that commercial kohl can contain more than 25 diff erent substances, some of which can provoke allergy; health problems have been caused by kohl containing lead. But we do not wish to issue a blanket warning against kohl. Ingredients of kohl vary widely. In south Asia, and across much of the Muslim world, traditional recipes are regarded as medicinal. Much-feared Pashtun warriors historically applied kohl before surging into battle.

Alien scale insects (Hemiptera Coccoidea) in European and Mediterranean countries: the fate of new and old introductions

This contribution focuses on recent interceptions and introductions of alien scale insects and their current distribution in European and Mediterranean countries. Data and collections were gathered in markets, nurseries, and botanical gardens, mostly in Italy, either indoors or outdoors. New or recent records of the following alien species are presented: Exallomochlus hispidus (Morrison); Ferrisia virgata (Cockerell) (Pseudococcidae); Coccus viridis (Green); Milviscutulus mangiferae (Green) (Coccidae); Aonidiella orientalis (Newstead); Aspidiotus destructor Signoret; Aulacaspis tubercularis Newstead; Fiorinia fioriniae Targioni Tozzetti; Lepidosaphes pinnaeformis (Bouché); Pseudaulacaspis brimblecombei Williams (Diaspididae). New data and pest status of Phoenicococcus marlatti Cockerell (Phoenicococcidae) and Trabutina mannipara (Hemprich & Ehrenberg) (Pseudococcidae) are also reported. The possible repeated introductions of the latter from North Africa to south Italy by trans-Mediterranean winds, is hypothesized.

Identification of pigmented Serratia marcescens symbiotically associated with Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae)

To characterize red pigment‐producing bacteria (RPPB) regularly released during oviposition by red palm weevil (RPW), RPPB were recovered from eggs deposited in apples supplied as substrate for oviposition. The presence of RPPB was also detected from gut, the reproductive apparatus of dissected adult and virgin insects and from pupal cases collected within infested palms. RPPB were also identified all along the tissue of these palms. Analysis of the 16S rDNA, gyrB, rpoB, recA, and groEL sequences assigned RPPB to the species Serratia marcescens. RPPB exhibited an antimicrobial activity assessed by the agar well diffusion method against a number of gram‐positive and gram‐negative bacteria. In this study, we first report the identification of a red pigment‐producing S. marcescens as extracellular symbiont of RPW. Route of transmission, detection within different organs, and a wide spread along the infested palm tissue, suggested S. marcescens is present as extracellular symbiont in different developmental stages of the RPW. Additionally, the antimicrobial activity exhibited versus Bacillus spp., Paenibacillus spp., and Lysinibacillus spp., reported as insect pathogens and potential candidates for biocontrol agents, could ascribe for S. marcescens a potential protective role.

Description of female nymphal instars and adult female of Kermes echinatus Balachowsky (Hemiptera, Coccoidea, Kermesidae) based on specimens from Crete and mainland Greece, with a discussion on geographical variation.

The first-instar nymph, second- and third-instar female nymphs and the adult female of Kermes echinatus Balachowsky (Hemiptera, Coccoidea, Kermesidae) are described and illustrated; micrographs of morphological details are also provided. The species was collected on the island of Crete (Greece) and on mainland Greece, new localities for this species, and are compared with Israeli specimens from where it was originally described.

Does Unaspis euonymi (Comstock) (Hemiptera: Diaspididae) host Serratia symbiotica Moran (Bacteria: Enterobacteriaceae)?

The euonymus scale Unaspis euonymi (Comstock) (Hemiptera: Diaspididae) is a pest of spindle that exhibits a strong preference for Euonymus, although it has been detected on at least 18 genera in 13 plant families (Buxus, Camellia, Celastrus, Daphne, Eugenia, Euonymus, Hibiscus, Ilex, Jasminum, Ligustrum, Lonicera, Olea, Pachistima, Pachysandra, Perychmenum, Prunus and Syringa) (Salisbury et al., 2013). Heavy infestation by this pest may lead to the death of the host plant and consequential loss of income from the cultivation of ornamental plants (Kaygin et al., 2008). U. euonymi is an armored scale insect originally from mild Eastern Asia and probably introduced into Europe in the 20th century (Pellizzari & Germain, 2010). Its lifecycle, depending on climate conditions, comprises two-three generations a year and the control measures to limit its diffusion mainly rely on the use of insecticides or the growing of resistant cultivars. The insects can engage mutualistic interactions or symbioses with a variety of bacteria that can profoundly affect the host’s biology. Apart from obligate symbionts (maternally transmitted), a growing number of facultative or secondary symbionts (that can be horizontally transmitted) have been identified (Sandstrom et al., 2001, Moran et al., 2008). Despite not being essential for the host’s life cycle, this last type of symbiont can strongly influence their fitness (Oliver et al., 2003, Jaenike & Brekke, 2011). Additionally, the mutualistic association between insects and bacteria may play a role in the evolution of the latter as described for some groups of Entereobacteriaceae (Moran et al., 2005). A number of genomic and phylogenetic studies on mutualistic associations between Enterobacteriaceae and aphids, psyllids, scale insects, whiteflies, weevils and other insects have been reported (Lefevre et al., 2004, Thao & Baumann, 2004). Here we report the identification of Serratia symbiotica (strain UESS2016) in U. euonymi adult females collected from Sofia (Bulgaria) in 2013.