The Culicoides Obsoletus Group In Italy: Relative .
The ‘Culicoides obsoletus group’ in Italy:relative abundance, geographic range, androle as vector for Bluetongue virusMaria Goffredo1*, Rudy Meiswinkel2, Valentina Federici1, Francesca Di Nicola1,Giuseppe Mancini1, Carla Ippoliti1, Alessio Di Lorenzo1, Michela Quaglia1, Adriana Santilli1,Annamaria Conte1 and Giovanni Savini112*Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Teramo, Italy.Entomologist, Santa Maria del Monte, Via Pretarone 14, Rocca di Cave, 00030 Rome, Italy.Corresponding author at: Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Campo Boario, 64100 Teramo, Italy.Tel.: 39 0861 332416, e-mail: m.goffredo@izs.it.Veterinaria Italiana 2016, 52 (3-4), 235-241. doi: 10.12834/VetIt.35.100.1Accepted: 03.06.2016 Available on line: 30.09.2016IV International Conference on Bluetongue and Related Orbiviruses. November 5‑7, 2014 ‑ Rome, Italy ‑ Selected papersKeywordsAvaritia,Culicoides chiopterus,Culicoides dewulfi,Obsoletus complex,Vector competence.SummaryAs Bluetongue virus (BTV) spread in Italy following its first incursion in 2000, it soon becameapparent that, besides Culicoides imicola, additional species of the subgenus Avaritiawere involved as vectors, namely one or more of the species that belong to the so-called‘Culicoides obsoletus group’, which comprises C. dewulfi, C. chiopterus, C. obsoletus sensustricto, C. scoticus and C. montanus; the three last named species are considered generally asforming the Obsoletus complex. This study presents the findings made over the last decadeand more, within the Italian entomological surveillance program for Bluetongue. It describesthe integrated morphological and molecular approach used to identify the species of the‘C. obsoletus group’, maps in detail their relative abundances and geographic ranges in Italy,clarifies the hitherto unknown comparative seasonal abundances of C. obsoletus s.s. andC. scoticus in a site in Central Italy, and provides further details on the potential vector statusof five species of the ‘C. obsoletus group’, with emphasis on C. obsoletus s.s., C. scoticus andC. montanus. Unlike the situation in Northern Europe, Culicoides dewulfi and C. chiopterus areuncommon to rare in Italy. In contrast, the Obsoletus complex occurs abundantly throughoutItaly, with C. obsoletus s.s. being the most prevalent and ecologically adaptive of the threespecies making up the complex. A longitudinal study conducted at a site in Central Italyrevealed that: (i) species of the Obsoletus complex prefer horses to sheep; (ii) their parity ratesrange from 10% (March) to 56% (November); (iii) throughout the year C. scoticus is consistentlymore abundant than C. obsoletus s.s.; (iv) abundances in both, C. obsoletus s.s. and C. scoticus,peak in May-June, with the peak of the latter species being more evident. Bluetongue viruswas first isolated from wild caught midges of the Obsoletus complex in 2002. Thereafter,pools of selected parous midges collected across Italy, and during multiple outbreaks of BT,have been found consistently PCR-positive for the virus. More recently, viral RNA has beendetected in field specimens of C. dewulfi, C. obsoletus s.s., C. scoticus and C. montanus.Il “Culicoides obsoletus group” in Italia:abbondanza, distribuzione geografica e ruolo vettorialeParole chiaveAvaritia,Competenza vettoriale,Culicoides chiopterus,Culicoides dewulfi,Obsoletus complex.RiassuntoSin dalle prime incursioni del virus della Bluetongue (BTV) in Italia a partire dal 2000, èrisultato evidente che altre specie di vettori del sottogenere Avaritia fossero coinvolte,oltre a Culicoides imicola, in particolare quelle del cosiddetto “Culicoides obsoletus group”,che comprende C. dewulfi, C. chiopterus, C. obsoletus sensu stricto, C. scoticus e C. montanus;le ultime tre specie formano nell’insieme l’Obsoletus complex. Questo lavoro riassume irisultati ottenuti in oltre dieci anni di sorveglianza entomologica per la BT. Descrive il metododi identificazione di specie, basato su un approccio integrato morfologico e molecolare,riporta le mappe di distribuzione e abbondanza delle singole specie, nonché l’abbondanzarelativa stagionale di C. obsoletus s.s. e C. scoticus in un sito di cattura permanente nell’Italia235
‘C. obsoletus group’ in ItalyGoffredo et al.Centrale, e fornisce informazioni sul possibile ruolo vettoriale in Italia di cinque specie del“C. obsoletus group”. Al contrario di quanto avviene in Nord Europa, Culicoides dewulfi eC. chiopterus non sono specie comuni in Italia. Invece l’Obsoletus complex è abbondantein tutta la penisola, soprattutto C. obsoletus s.s., che sembra infatti essere la specie piùadattabile. Uno studio longitudinale, condotto in un sito permanente di cattura nell’Italiacentrale, ha evidenziato che: (i) le specie dell’Obsoletus complex preferiscono i cavallialle pecore; (ii) la percentuale dei parous nella popolazione varia dal 10% (marzo) al 56%(novembre); (iii); C. scoticus è costantemente più abbondante di C. obsoletus s.s., durantetutto l’anno; (iv); entrambi C. obsoletus s.s. e C. scoticus mostrano un picco di abbondanzain maggio-giugno, il secondo con un picco più marcato. Il BTV è stato isolato per la primavolta in Italia dall’Obsoletus complex nel 2002, mentre successivamente vari sierotipi sonostati rilevati tramite PCR, nel corso delle diverse epidemie di BT, recentemente, RNA viraleè stato riscontrato in campo, per la prima volta in Italia, da C. dewulfi, e inoltre da pool diC. obsoletus s.s., C. scoticus e C. montanus, identificati a livello specifico.IntroductionHistorically, the year 2000 marks the first incursionof Bluetongue (BT) into Italy. The disease emerged insheep and spread through the principal Afro‑Asianvector Culicoides imicola Kieffer on Sardinia island.In 2001, Bluetongue reached the mainland andsoon began to spread into areas where C. imicolawas absent; in time this led to the discovery thatalso other biting midge species act as vectors forBluetongue virus (BTV), namely Culicoides obsoletus(Meigen) and Culicoides scoticus Downes and Kettle(Savini et al. 2005). In the decade since its arrivalin the Mediterranean region, BTV has continuedto incur and spread ever northwards, eventuallyreaching 58 N, more than 1,000 km beyond itstraditional limit of 45 N. Simultaneously, other bitingmidge species have been added to the growingvector list and now includes also Culicoides dewulfiGoetghebuer, Culicoides chiopterus (Meigen) andCulicoides montanus Shakirzjanova.All six of the abovementioned species belong to thesubgenus Avaritia Fox; excepting for C. imicola, mostresearchers consider the five remaining speciesto form the ‘C. obsoletus group’. Phylogeneticallyspeaking, this group does not form a natural(monophyletic) group so, cladistically speaking,is artificial. Based on adult morphology, onlyC. obsoletus, C. scoticus and C. montanus form anatural group, the Obsoletus complex (not to beconfused with the ‘C. obsoletus group’); this narrowinterpretation has the effect of leaving C. chiopterusand C. dewulfi as two separate, free‑floatingmonotypic entities within Avaritia. To a greatextent, this fragmented arrangement is supportedmolecularly, but it remains unclear as to whetherC. chiopterus should be included (Schwenkenbacheret al. 2009, Schulz et al. 2015) or excluded(Meiswinkel et al. 2004, Gomulski et al. 2005, Anderet al. 2013) from the Obsoletus complex. Ultimately,236the phylogeny of the subgenus Avaritia will beresolved only once the entire Holarctic fauna (ofapproximately 30 species) is treated as a whole.In the interim, we will continue to use theparaphyletic taxon ‘C. obsoletus group’, but retain itin double quotes to signpost its cladistic artificiality.In Europe today, the ‘C. obsoletus group’ (excludingC. imicola) comprises seven species; the actualnumber is probably closer to ten. As noted above,five species of the ‘C. obsoletus group’ occur inItaly, namely C. obsoletus sensu stricto, C. scoticus,C. montanus, C. dewulfi and C. chiopterus. Integratedmorphological and molecular studies indicate theoccurrence of a fourth species of the Obsoletuscomplex in Italy and referred to provisionally as‘Culicoides sp. unidentified’ (Gomulski et al. 2005); itdoes not form part of this study.Below, with reference to the ‘C. obsoletus group’, wepresent a brief five‑part review of the entomologicalfindings made in Italy since 2000 and obtained underthe aegis of the Italian entomological surveillanceprogram for Bluetongue:i. brief overview (above) of the unresolvedtaxonomy of the ‘C. obsoletus group’;ii. description of the integrated morphologicaland molecular approach used within theItalian entomological surveillance programof Bluetongue to identify species of the‘C. obsoletus group’;iii. mapping of the relative abundances andgeographic ranges of each species in Italy;iv. clarifying the hitherto unknown comparativeseasonal abundances of C. obsoletus s.s. andC. scoticus based on a longitudinal studyconducted at a site in Central Italy, andv. the potential vector status of each of the fivespecies of the ‘C. obsoletus group’ is detailed,Veterinaria Italiana 2016, 52 (3-4), 235-241. doi: 10.12834/VetIt.35.100.1
Goffredo et al.‘C. obsoletus group’ in Italywith emphasis on C. obsoletus s.s., C. scoticusand C. montanus.Culicoides species, and utilizes in combination fourwing pattern characters (Goffredo and Meiswinkel,2004), which must be applied strictly:Integrated morphological andmolecular approach used to identifyspecies of the ‘C. obsoletus group’ distal part of second radial wing cell (r2)partially pale;Various approaches are used to identify individualspecies of the C. obsoletus group. These includetraditional morphology, advanced morphometrics,mass spectrometry, various polymerase chainreaction (PCR) tests, microarrays, and the molecularsequencing of specific gene regions, in particularCO1 (barcoding), ITS1 and ITS2. Within the Italiansurveillance programme for Bluetongue, thelaboratory analysis of an entomological field sampleis treated as rigorously as other kinds of field samplesare (and involving either blood, serum, or animaltissue). Furthermore, vector species identification,which requires the screening of hundreds ofsamples monthly, needs to be achieved rapidlyand accurately. For these reasons, a standardizedprotocol, integrating both morphological andmolecular methods, has been developed, not onlyto differentiate between cryptic species such asC. obsoletus and C. scoticus, but also to confirmthe identity of rarer species (such as C. dewulfi andC. chiopterus) (Figure 1). pale wing spots mostly diffuse, wing bisectedinto a paler basal half and a darker distal half;pale spot in the apex of r5 poorly defined,round or square, andThe first step, conducted under the dissectingmicroscope, involves the morphological separationof specimens of the ‘C. obsoletus group’ from other wing macrotrichia sparse, restricted to distalhalf or third of wing; absence of a central dark spot in the cubitalwing cell.The females of C. chiopterus and C. dewulfi can bedistinguished from each other, and from speciesof the Obsoletus complex, based on morphology(Campbell and Pelham‑Clinton 1960, Delécolle1985). However, it is far more difficult to identifythe females of the three (or more) sibling speciesof the Obsoletus complex and is the reason whyspecies‑specific molecular assays have beendeveloped over the last decade and are being usedincreasingly.Morphologically, C. dewulfi is the only species ofAvaritia in Europe in which the female has 8 longsetae projecting laterally from the first abdominalsegment; all the remaining species possess fewersetae, usually in the range of 2‑5. Furthermore, inC. dewulfi, the two spermathecae differ significantlyin size, while the wing has a more rounded shape;‘Culicoides obsoletus group’C. chiopterus, C. dewulfi, Obsoletus ComplexC. chiopterus Very pale wing Hairy eyes Smaller size Possible confirmationby RT-PCR based on ITS-1Obsoletus complex(C. obsoletus, C. scoticus, C. montanus) Multiplex RT-PCR based on ITS-2(positive control)C. scoticusC. montanus(positive control)(positive control)C. obsoletusmultiplex RT-PCR for the identification of C. obsoletus (O), C. scoticus (S) and C. montanus (M)400 bp213 bpC. dewulfi89 bpMSOOSRT-PCR for the identification of C. dewulfiC. dewulfiO(positive control) Shape of pale spot in the apex of r5 Asymmetric spermathecae 8 to 12 long lateral hairs on the firstabdominal segment Possible confirmationby RT -PCR based on ITS2252 bp294 bpFigure 1. Integrated morphological and molecular approach to identify the species of the ‘C. obsoletus group’.Veterinaria Italiana 2016, 52 (3-4), 235-241. doi: 10.12834/VetIt.35.100.1237
‘C. obsoletus group’ in ItalyGoffredo et al.unfortunately, both characters are very difficultto observe and can be quantify only under adissecting microscope with very high resolution.Culicoides chiopterus is one of the smallest EuropeanCulicoides species, however size is not definitive aslarger specimens do exist and will form part of anycollection made. Uniquely, the wing in C. chiopterus isyellowish rather than greyish; furthermore, the palespots are very poorly defined and difficult to observe,whilst on the wing the macrotrichia are sparse andrestricted to the distal third. However, the near lack ofa well‑defined wing pattern means that C. chiopterusmay be misidentified as a plain‑wing species.However, the 10 plain‑winged species that make upthe European fauna, all differ from C. chiopterus inhaving the wing noticeably more ‘hairy’, i.e. the entirewing is covered densely with macrotrichia and, inmost species, are dark and easy to observe.A practiced eye is however needed to identifyaccurately species of Avaritia based on morphologyalone, especially in the female, the sex that formsthe bulk ( 95%) of any light‑trap collection made.Especially time‑consuming, is to morphologicallydifferentiate between C. obsoletus s.s. and C. scoticusand which occur widely and abundantly across Italy.Consequently, while assaying Culicoides for thepresence of virus, it is becoming the norm to identifythem simultaneously to species molecularly.Within the protocol used for the Italian BTentomological surveillance, an in‑house multiplexreal time polymerase chain reaction (RT‑PCR)has been developed to differentiate betweenAC. obsoletus s.s., C. scoticus and C. montanus; it is basedon the internal transcribed spacer 2 (ITS2) (Gomulskiet al. 2005). An additional species‑specific RT‑PCR,based also on ITS2, was developed to confirm theidentification of C. dewulfi. Control plasmids areused as positive reaction controls. These PCRs arebased on field samples of C. obsoletus s.s. (male),C. scoticus (male), C. dewulfi and C. montanus,their identities based on homologous sequencesavailable in GenBank; to confirm the identificationof C. chiopterus, an RT‑PCR based on ITS1, is used(Mathieu et al. 2007).It is important to conclude by saying that within theHolarctic region, the taxonomy of the ‘C. obsoletusgroup’ is not resolved. At least three cryptic species– related closely to the five listed above and notforming part of current molecular assays – areknown to exist (Gomulski et al. 2005, Meiswinkelet al. 2004, Meiswinkel et al. 2015, Kirkeby et al. 2014,Ander et al. 2012).Distribution and abundanceBased on 6,000 light‑trap collections made acrossItaly, the geographic range and seasonal abundancesof the species that comprise the ‘C. obsoletus group’have been clarified; the three species that comprisethe Obsoletus complex (C. obsoletus s.s., C. scoticusand C. montanus) are mapped separately, based on3,020 individual midges – selected randomly andidentified molecularly – from 87 sites (Figure 2).BRegionsObsoletus complex1-910 - 99100 - 9991000 - 43362Species (%)Obsoletus s.s.C. scoticusC. montanusRegionsFigure 2. Abundance and distribution map of the Obsoletus complex (A), and relative abundance of C. obsoletus, C. scoticus and C. montanus in Italy (B).238Veterinaria Italiana 2016, 52 (3-4), 235-241. doi: 10.12834/VetIt.35.100.1
Goffredo et al.‘C. obsoletus group’ in ItalyRegionsCulicoides dewulfi01 - 1011 - 5051 - 150151 - 1890RegionsCulicoides chiopterus01-910 - 102Figure 4. Abundance and distribution map of C. chiopterus in Italy.Figure 3. Abundance and distribution map of C. dewulfi in Italy.Compared to C. imicola, the Obsoletus Complexfavours more densely vegetated habitats withincreased green leaf density (Conte et al. 2007).Within the Obsoletus complex, although bothC. obsoletus s.s. and C. scoticus are usually presentat a collection site, the former is the most prevalentand ecologically adaptive of the three species thatmake up the Obsoletus complex.A statistical Bayesian approach (Beta distribution)was used to assign the sites to a prevalent speciesand resulted in 52 sites for C. obsoletus s.s., eight forC. scoticus and three for C. montanus. Landscapeand remote sensing analysis, applied to these sites(Ippoliti et al. 2016) showed that compared toC. obsoletus s.s., which is often encountered withinurban and cultivated areas, C. scoticus is morehabitat selective, favouring naturally vegetatedareas, including forests. Finally, C. montanus is by farthe rarest of the three species and is found mainlyat lower altitudes in the drier southern half of Italy.Veterinaria Italiana 2016, 52 (3-4), 235-241. doi: 10.12834/VetIt.35.100.1i.species of the Obsoletus complex preferhorses to sheep (100 paired collections,U‑Mann Whitney test 43.47, p 0.001);ii. based on 341 collections made near to horsesbetween April 2003 and March 2007, adultfemales were active throughout the year, theirparity rates ranging from 10% (March) to 56%(November) (Figure 5);iii. based on at least 20 nulliparous females(identified by multiplex PCR) extracted atrandom from the 15 largest collections mademonthly between January 2006-March 2007, itwas determined that the relative abundancesNo. midgesCulicoides chiopterus is rarely captured in Italy, withC. dewulfi uncommon. Although C. dewulfi occursthroughout the length of the mainland, it rarelyexceeds 3% of any light‑trap collection made nearlivestock; the record catch is 1,890 specimens (relativeabundance 21,7%) (Figures 3 and 4). In contrast, theObsoletus complex, in particular C. obsoletus s.s.and C. scoticus, occurs abundantly around livestockand throughout the whole of Italy. In NorthernItaly, C. obsoletus s.s. is the most prevalent, by far. Insharp contrast, C. montanus – the third species of theObsoletus complex – is very rare (Goffredo et al. 2013).A longitudinal light‑trap study conducted at apermanent collection site in Central Italy 00040000200000Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec(25.1) (19.7) (9.6) (27.0) (29.3) (46.3) (42.8) (39.0) (35.8) (32.3) (55.6) (31.5)Month(parity rate %)NulliparousEngorgedParousTotal Obsoletus complexFigure 5. Age grading and parity rate of the Obsoletus complex ina permanent collection site in Central Italy. Total number of midgesper month has been considered, from 341 collections near horses,between April 2003 and March 2007.239
‘C. obsoletus group’ in ItalyAGoffredo et al.Table I. Vector status of the ‘C. obsoletus group’ in Italy, for Bluetonguevirus (BTV) and related orbiviruses (African horse sickness virus, AHSV,and Epizootic haemorrhagic disease virus, EHDV).100%80%1360%153026 25261741434383820261240%20%751014 151426142266810Jan0Fe 6bM 06ar-0Ap 6r-0M 6ay-0Ju 6n0Ju 6l-0Au 6g0Se 6p0Oc 6t-0No 6v0De 6c06Jan0Fe 7bM 07ar-070%20Month/yearLog midgesBC. obsoletus54.543.532.521.510.50C. scoticus06 06 06 06 06 06 06 06 06 06 06 06 07 07 07n- eb- ar- pr- ay- un- Jul- ug- ep- ct- ov- Dic- an- eb- arJ F MJF M A MS O NAMonth/yearFigure 6. Seasonal abundance of C. obsoletus and C. scoticus ina permanent collection site in Central Italy, basing on 15 biggestcollection/month (January 2006 - Mar
IV International Conference on Bluetongue and Related Orbiviruses. November 5‑7, 2014 ‑ Rome, Italy ‑ Selected papers Keywords Avaritia, Culicoides chiopterus, Culicoides dewulfi, Obsoletus complex, Vector competence. Veterinaria Italiana 2016, 52 (3-4), 235-241. doi: 10.12834/VetIt.35.100.1 Accepted: 03.06.2016 Available on line: 30.09.2016 Parole chiave Avaritia, Competenza .
May 02, 2018 · D. Program Evaluation ͟The organization has provided a description of the framework for how each program will be evaluated. The framework should include all the elements below: ͟The evaluation methods are cost-effective for the organization ͟Quantitative and qualitative data is being collected (at Basics tier, data collection must have begun)
Silat is a combative art of self-defense and survival rooted from Matay archipelago. It was traced at thé early of Langkasuka Kingdom (2nd century CE) till thé reign of Melaka (Malaysia) Sultanate era (13th century). Silat has now evolved to become part of social culture and tradition with thé appearance of a fine physical and spiritual .
IV International Conference on Bluetongue and Related Orbiviruses. November 5‑7, 2014 ‑ Rome, Italy ‑ Selected papers Parole chiave Culicoides spp., Moscerino, Sudafrica, Svernamento, Virus della Bluetongue, Virus della Peste equina africana. Riassunto I Culicoides sono noti vettori del virus della Bluetongue (BTV) e del virus della Peste equina africana (AHSV). Il lavoro riporta i .
On an exceptional basis, Member States may request UNESCO to provide thé candidates with access to thé platform so they can complète thé form by themselves. Thèse requests must be addressed to esd rize unesco. or by 15 A ril 2021 UNESCO will provide thé nomineewith accessto thé platform via their émail address.
̶The leading indicator of employee engagement is based on the quality of the relationship between employee and supervisor Empower your managers! ̶Help them understand the impact on the organization ̶Share important changes, plan options, tasks, and deadlines ̶Provide key messages and talking points ̶Prepare them to answer employee questions
Dr. Sunita Bharatwal** Dr. Pawan Garga*** Abstract Customer satisfaction is derived from thè functionalities and values, a product or Service can provide. The current study aims to segregate thè dimensions of ordine Service quality and gather insights on its impact on web shopping. The trends of purchases have
Chính Văn.- Còn đức Thế tôn thì tuệ giác cực kỳ trong sạch 8: hiện hành bất nhị 9, đạt đến vô tướng 10, đứng vào chỗ đứng của các đức Thế tôn 11, thể hiện tính bình đẳng của các Ngài, đến chỗ không còn chướng ngại 12, giáo pháp không thể khuynh đảo, tâm thức không bị cản trở, cái được
Organization Behavior is not a /an a. A separate field of study b. Applied science c. Normative science d. Pessimistic approach 13. “Cognitive theory” of learning was given by a. Skinner b. Pavlov c. Tolman d. Piajet 14. Extension of behavior modification into organization is called a. Enrichment b. Enlargement c. OB Mod d. OB Ext 15. -----is a relatively permanent change in behavior that .
