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Cited by 61Parasitic Diseases Research and TreatmentResearch on Leishmaniasis StudiesHelminth infection and control

First molecular identification of mosquito vectors of Dirofilaria immitis in continental Portugal

Open access
Ferreira, Cátia Alexandra Costa, de Pinho Mixão, Verónica, Novo, Maria Teresa Lourenço Marques, Calado, Maria Manuela Palmeiro, Gonçalves, Luzia Augusta Pires, Belo, Silvana Maria Duarte, de Almeida, António Paulo Gouveia·2015·Parasites & Vectors
DOI 10.1186/s13071-015-0760-2PMC PMC4369802
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Abstract

BACKGROUND: Canine dirofilariasis due to Dirofilaria immitis is known to be endemic in continental Portugal. However, information about the transmitting mosquito species is still scarce, with only Culex theileri identified to date, albeit with L1-2, through dissection. This study was carried out to investigate the potential vectors of Dirofilaria spp. in continental Portugal. METHODS: Mosquitoes were collected in three distinct seasons (Summer, Autumn and Spring), 2011-2013, in three districts. CDC traps and indoor resting collections were carried out in the vicinity of kennels. Mosquitoes were kept under controlled conditions for 7 days to allow the development of larval stages of Dirofilaria spp.. DNA extraction was performed separately for both head+thorax and abdomen in order to differentiate infective and infected specimens, respectively, in pools, grouped according to the species and collection site (1-40 specimen parts/pool), and examined by PCR using pan-filarial specific primers. Mosquito densities were compared using non-parametric tests. Dirofilaria development units (DDU) were estimated. RESULTS: In total, 9156 female mosquitoes, from 11 different species, were captured. Mosquito densities varied among the 3 districts, according to capture method, and were generally higher in the second year of collections. From 5866 specimens screened by PCR, 23 head+thorax and 41 abdomens pools, corresponding to 54 mosquitoes were found positive for D. immitis DNA. These belonged to 5 species: Culex (Cux) theileri (estimated rate of infection (ERI)=0.71%), Cx. (Cux) pipiens f. pipiens and f. molestus (ERI=0.5%), Anopheles (Ano) maculipennis s.l. (ERI=3.12%), including An. (Ano) atroparvus, Aedes (Och) caspius (ERI=3.73%) and Ae. (Och) detritus s.l. (ERI=4.39%). All but Cx. pipiens, had at least one infective specimen. No D. repens infected specimens were found. Infection rates were: 3.21% in Coimbra, 1.22% in Setúbal and 0.54% in Santarém. DDU were at least 117/year in the study period. CONCLUSIONS: Culex theileri, Cx. pipiens, An. maculipennis s.l. An. atroparvus, Ae.caspius and Ae. detritus s.l. were identified as potential vectors of D. immitis in three districts of Portugal, from Spring to Autumn, in 5 of the 6 collection dates in 2011-2013. Implications for transmission, in the context of climate changes, and need for prophylactic measures, are discussed.

AI snapshot

Written from the paper’s full text.

TL;DR

This study provides the first molecular evidence of Dirofilaria immitis in mosquitoes from continental Portugal, identifying multiple potential vector species across three districts. It reports infected and infective mosquitoes from Spring to Autumn and estimates 117–152 favorable transmission days per year.

Key findings

  • From 9,156 female mosquitoes (11 species), 5,866 were PCR-screened; 54 positive pools/mosquitoes were found for D. immitis DNA, including 23 head+thorax and 41 abdomen pools.
  • Estimated infection rates were Cx. theileri 0.71%, Cx. pipiens f. pipiens/f. molestus 0.5%, An. maculipennis s.l. 3.12% (including An. atroparvus), Ae. caspius 3.73%, and Ae. detritus s.l. 4.39%; all except Cx. pipiens had at least one infective specimen.
  • District infection rates were Coimbra 3.21%, Setúbal 1.22%, and Santarém 0.54%, differing significantly overall (χ2=40.93, df=2, P<0.0001).
  • No D. repens-infected specimens were found; DDU estimates indicated at least 152 days in 2011, 119 in 2012, and 117 in 2013 suitable for extrinsic development.

Methods

  • Mosquitoes were collected in 2011–2013 during Summer, Autumn, and Spring in Coimbra, Santarém, and Setúbal using CDC light traps with dry ice and indoor resting aspirator collections near kennels; specimens were kept 7 days at 25±2°C, 70±5% RH, 12 h:12 h light:dark, and fed 10% sucrose.
  • Females were dissected into head+thorax and abdomen; DNA was extracted by CTAB and screened by pan-filarial PCR (DIDR-F1/R1) in pools of 1–40 body parts; Cx. pipiens positives were identified to form using CQ11 and ace-2 PCR, and products were sequenced/BLAST-confirmed.
  • Infection rates were estimated as MIR and ERI; mosquito densities were compared with non-parametric tests (Mann–Whitney, Kruskal–Wallis, Dunn–Bonferroni), and Dirofilaria development units (DDU) were calculated from temperatures >14°C and DDU30 ≥130.

Limitations

  • The paper states that infection rates based on sample sizes <1,000 may not accurately represent true population infection rates; An. maculipennis s.l. (n=400) and Ae. caspius (n=270) had large 95% CIs, and Ae. detritus s.l. (n=23) had a very wide CI, reducing the significance of its high rate.
  • The paper states that only three collecting moments per year were insufficient to draw conclusions on seasonal mosquito dynamics, and that the 7-day insectary period at 25°C may not allow complete extrinsic cycle development, being a compromise for mosquito mortality, DNA degradation, and bloodmeal digestion.
  • Evident from the text: PCR detects D. immitis DNA rather than confirming viable L3, although head+thorax positivity was used as an infective proxy; Ae. detritus s.l. was not differentiated due to available technique; and Setúbal densities were lower than in previous surveys, possibly due to kennel-site collection design, few collections, and strong winds.

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Integrity signals

Registry

Crossref · 10.1186/s13071-015-0760-2

Cites

51 of 51 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 62 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Of 5866 mosquitoes screened by PCR, 54 were positive for D. immitis DNA (23 head+thorax pools and 41 abdomen pools).

    “From 5866 specimens screened by PCR, 23 head+thorax and 41 abdomens pools, corresponding to 54 mosquitoes were found positive for D. immitis DNA.”
    Quote in the paperSubstantial claim
  • Findingown data

    The mosquito infection rate in Coimbra (3.21%) was significantly higher than in Setúbal (1.22%).

    “or Setúbal (1.22%, χ2=19.46, Df=2, P<0.0001)”
    Quote in the paperNot a substantial claim
  • Findingown data

    D. immitis DNA was detected in Ae. detritus s.l. with an ERI of 4.39%.

    “| Ae. detritus s.l. | 23 | 23 | 16 | 1 | 1 | 1 | 43.5/1000 | 4.39% | 0.008–0.21 |”
    Quote in the paperSubstantial claim
  • Findingown data

    In 2011, there were at least 152 days with suitable conditions for the completion of the extrinsic development of Dirofilaria spp. in the three studied districts.

    “there was, at least, 152 days in 2011, 119 days in 2012 and 117 days in 2013 with suitable conditions for the completion of the extrinsic development of Dirofilaria spp.”
    Quote in the paperSubstantial claim
  • Findingown data

    The ERI of D. immitis in mosquitoes in Setúbal was 1.22%.

    “| Setúbal | 659 | 658 | 398 | 1 | 8 | 8 | 12.2/1000 | 1.22% | 0.006–0.024 |”
    Quote in the paperNot a substantial claim
  • May combine two claims. 22
  • Classified as probably not a substantial claim. 31

  • May combine two claims.

    Findingown data

    The PCR assay detected parasite DNA at a 1:40 dilution, corresponding to one positive mosquito in a pool of 40, for both head+thorax and abdomen pools.

    “Parasite DNA was detected until 1:40 dilution, either for head+thorax or abdomen pools, corresponding to one single positive mosquito in a pool of 40 mosquitoes.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Mosquitoes with D. immitis DNA were found in all collecting dates except November 2011, usually by both methods and in more than one district.

    “Overall, mosquitoes with D. immitis DNA were found in all collecting dates, but November 2011, usually by both methods and in more than one district (Figure 2, Table 5).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Cx. pipiens s.l. positive pools for D. immitis were identified as Cx. pipiens s.s., 7 form pipiens and 1 form molestus, with the molestus from Setúbal.

    “Culex pipiens s.l. positive pools for D. immitis, were identified as Cx. pipiens s.s., 7 form pipiens and 1 form molestus, which was from Setúbal.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    DDU were at least 117/year in the study period.

    “DDU were at least 117/year in the study period.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    D. immitis DNA was found in the four most frequent mosquito species, and also in Ae. detritus s.l.

    “Dirofilaria immitis DNA was found in the four most frequent species, but also in Ae. detritus s.l. (Table 3, with respective values of MIR, ERI and 95% CI).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    D. immitis DNA was detected in Cx. theileri with an estimated rate of infection (ERI) of 0.71%.

    “| Cx. theileri | 5812 | 3406 | 234 | 13 | 16 | 23 | 6.8/1000 | 0.71% | 0.005–0.01 |”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For An. maculipennis s.l. and Ae. caspius, the sample sizes were 400 and 270, respectively, with somewhat large 95% CIs.

    “in the case of An. maculipennis s.l. and Ae. caspius the sample size is 400 and 270, respectively, with somewhat large 95% CIs”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    A great proportion of the pools were of a single mosquito specimen.

    “probably as a great proportion of our pools were of a single mosquito specimen.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    For Ae. detritus s.l., the sample size was 23 specimens with a much wider 95% CI.

    “in the case of Ae. detritus s.l., with a sample of 23 specimens and a much wider 95% CI”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The species with the highest infection rates were Ae. caspius (3.7%), An. maculipennis s.l. (3.1%), Cx. theileri (0.7%), and Cx. pipiens s.l. (0.5%).

    “The species with the highest infection rates were Ae. caspius (3.7%), followed by An. maculipennis s.l. (3.1%), Cx. theileri (0.7%) and Cx. pipiens s.l. (0.5%).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Highest and lowest mosquito infection rates were registered in Coimbra and Santarém, respectively.

    “Highest and lowest infection rates for mosquitoes were registered in Coimbra and Santarém, respectively”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Cx. pipiens s.l. specimens were individually analyzed because of the sympatric existence of the two biological forms pipiens and molestus in Portugal.

    “Specimens of Cx. pipiens s.l. were individually analyzed, due to the sympatric existence of the two biological forms, pipiens and molestus of the sensu strictu species, in Portugal [30].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [30] Gomes B, Sousa C, Novo M, Freitas FB, Alves R, Côrte-Real AR, et al. Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region. Portugal BMC EvoBiol. 2009;9:262.

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    There is no extrinsic development of D. immitis in the mosquito at average temperatures ≤14°C.

    “For each day in which the average temperature was >14°C, temperature at which there is no extrinsic development of the parasite, the difference between the average temperature and 14°C was calculated (i.e. for Tmean≥15, DDU=Tmean-14) [40].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [40] Fortin JF, Slocombe JOD. Temperature requirements for the development of Dirofilaria immitis in Aedes triseriatus and Ae. vexans. Mosq News. 1981;41:625–33.

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Mosquito females were dissected into head+thorax and abdomen to discriminate between infective and infected Dirofilaria status, respectively.

    “Mosquito females were dissected into head+thorax and abdomen to discriminate between Dirofilaria spp. infective/infected status, respectively [29].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [29] Favia G, Lanfrancotti A, Della Torre A, Cancrini G, Coluzzi M. Polymerase chain reaction identification of Dirofilaria repens and Dirofilaria immitis. Parasitology. 1996;6:567–71. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In 2009-2010, the overall seroprevalence of canine dirofilariasis in Northern and Central Portugal was 2.1%.

    “In 2009–2010 the overall sero-prevalence in Northern and Central Portugal was 2.1% for CD [23].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] Vieira L, Silvestre-Ferreira AC, Fontes-Sousa AP, Balreira AC, Morchón R, Carretón E, et al. Seroprevalence of heartworm (Dirofilaria immitis) in feline and canine hosts from central and northern Portugal. JournHelminth. 2014;14:1–5.

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In 2011-2013, the overall parasitological prevalence of canine dirofilariasis in three districts of Centre-South Portugal was 15.1%.

    “A recent survey, 2011–2013, in three districts of Centre-South, has revealed an overall parasitological prevalence rate of 15.1%, the highest in Setúbal (24.8%), followed by Coimbra (13.8%) and Santarém (13.2%) [24].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [24] Alho AM, Landum M, Ferreira C, Meireles J, Gonçalves L, Madeira de Carvalho L etal. Prevalence and seasonal variations of canine dirofilariosis in Portugal. Vet Parasitol 2014, doi:10.1016/j.vetpar.2014.08.014. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Dirofilaria spp. are transmitted by several mosquito species belonging to a wide range of genera in different parts of the world, such as Culex, Aedes and Anopheles.

    “Dirofilaria spp. are transmitted by several mosquito species belonging to a wide range of genera in different parts of the world, such as Culex, Aedes and Anopheles [5].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] Simón F, Siles-Lucas M, Morchón R, González-Miguel J, Mellado I, Carretón E, et al. Human and animal dirofilariasis: the emergence of a zoonotic mosaic. ClinMicrobiol Rev. 2012;25:507–44.

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Culex theileri, Cx. pipiens, An. maculipennis s.l., An. atroparvus, Ae. caspius, and Ae. detritus s.l. are potential vectors of D. immitis in three districts of Portugal.

    “Culex theileri, Cx. pipiens, An. maculipennis s.l. An. atroparvus, Ae.caspius and Ae. detritus s.l. were identified as potential vectors of D. immitis in three districts of Portugal, from Spring to Autumn, in 5 of the 6 collection dates in 2011–2013.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    High densities of Cx. theileri, Cx. pipiens s.l., An. maculipennis s.l., and Ae. caspius were recorded in the study areas.

    “In addition, high densities of mosquito populations, namely Cx. theileri, Cx. pipiens s.l. An. maculipennis s.l. and Ae.caspius, were recorded in the above mentioned areas [26].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Almeida APG, Galão RP, Sousa CA, Novo MT, Parreira R, Pinto J, et al. Potential mosquito vectors of arboviruses in Portugal: species, distribution, abundance and West Nile infection. Transact Royal Soc Trop Med Hygiene. 2008;102:823–32. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Transmission of dirofilariasis depends on sufficient numbers of infected and microfilaremic dogs, susceptible mosquitoes, and a suitable climate for extrinsic incubation of the parasite.

    “Transmission of dirofilariasis is dependent upon the presence of sufficient numbers of infected and microfilaremic dogs, susceptible mosquitoes, and a suitable climate to allow extrinsic incubation of the parasite in the mosquito vector [13,14].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [13] Genchi C, Rinaldi L, Mortarino M, Genchi M, Cringoli G. Climate and Dirofilaria infection in Europe. Vet Parasitol. 2009;163:286–92. source

      Not checked in the free run.

    • cites [14] Medlock LM, Barras I, Kerrod E, Taylor MA, Leach S. Analysis of climatic predictions for extrinsic incubation of Dirofilaria in the United Kingdom. Vector Borne Zoon Dis. 2007;7:4–14. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In Portugal, there are records of Cx. pipiens and Cx. torrentium as members of the Cx. pipiens complex.

    “In Portugal, there are to date, records of Cx. pipiens and Cx. torrentium as members of the Cx. pipiens complex [46].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [46] Ribeiro H, Ramos HC, Pires CA, Capela RA. An annotated checklist of the mosquitoes of continental Portugal (Diptera, Culicidae) . Actas do III Congresso Ibérico de Entomologia [Proceedings of the III Iberian Congress of Entomology], 1988, 233–254.

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Cx. torrentium is rare and occurs only in northern and mountain areas of Portugal.

    “Culex torrentium is rare and occurs only in northern and mountain areas of the country [46]”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [46] Ribeiro H, Ramos HC, Pires CA, Capela RA. An annotated checklist of the mosquitoes of continental Portugal (Diptera, Culicidae) . Actas do III Congresso Ibérico de Entomologia [Proceedings of the III Iberian Congress of Entomology], 1988, 233–254.

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In Portugal, previous detection of Dirofilaria spp. L1 and L2 larvae in the Malpighian tubules of Cx. theileri in Setúbal yielded an infection rate of 4.76% in a sample of 42.

    “In Portugal, previous detection of Dirofilaria spp. L1 and L2 larvae in the MT of Cx. theileri, in the district of Setúbal, had yielded an infection rate of 4.76%, again, in a small sample (N=42) [25].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Ribeiro H, Ramos HC, Pires CA. Contribuição para o estudo dos vectores de filaríases animais em Portugal. J Socied Ciências Médicas. 1983;147:143–6.

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    An. maculipennis s.l. has previously been found infected with D. immitis.

    “Anopheles maculipennis s.l. has also been previously found infected with D. immitis [16].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Cancrini G, Magi M, Gabrielli S, Arispici M, Tolari F, Dell’Omodarme M, et al. Natural vectors of dirofilariasis in rural and urban areas of the Tuscan region, central Italy. J Med Entomol. 2006;43:574–9. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Cx. pipiens form pipiens and form molestus are likely vectors of D. immitis, based on detection of D. immitis DNA only in their abdomens and absence of bloodmeal traces.

    “and Cx. pipiens form pipiens and form molestus, as likely vectors, i.e.with DNA only in abdomens, but from mosquitoes without any traces of bloodmeal.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Cx. quinquefasciatus has not been recorded in Portugal.

  • May combine two claims.

    Premisecitation

    Infection rates in Ae. caspius in Italy were 0.18%, much lower than in this study.

    “Infection rates in Ae. caspius in Italy were much lower than ours, 0.18% [17].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Latrofa MS, Montarsi F, Ciocchetta S, Annoscia G, Dantas-Torres F, Ravagnan S, et al. Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis. Parasites Vectors. 2012;5:76. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In Italy, D. repens has been found in Cx. pipiens and Ae. albopictus at similar rates as D. immitis.

    “or at similar rates as D. immitis in Cx. pipiens and Ae. albopictus [44]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] Cancrini G, Scaramozzino P, Gabrielli S, di Paolo M, Toma L, Romi R. Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in central Italy. J Med Entomol. 2007;44:1064–6. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ae. detritus s.l. had been found positive for D. immitis in the abdominal portion.

    “and Ae. detritus s.l. for the abdominal portion [45]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] Cancrini G, Gabrielli S. Vectors of Dirofilaria nematodes: biology, behaviour and host/parasite relationships. In: Genchi C, Rinaldi L, Cringoli G, editors. Dirofilariaimmitis and D. repens in dog and cat and human infection. Naples: Rolando editore; 2007. p. 49–58.

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A historical study considered Cx. theileri as a probable vector of Dirofilaria spp.

    “with a historical study considering Cx. theileri as a probable vector of Dirofilaria spp. [25]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Ribeiro H, Ramos HC, Pires CA. Contribuição para o estudo dos vectores de filaríases animais em Portugal. J Socied Ciências Médicas. 1983;147:143–6.

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    An. maculipennis s.l., Ae. caspius, Ae. detritus s.l., and Cx. theileri are likely competent vectors of D. immitis, based on detection of D. immitis DNA in their head+thorax.

    “We report the finding of An. maculipennis s.l., Ae. caspius, Ae. detritus s.l. and Cx. theileri as likely competent vectors of D. immitis, i.e. with DNA in head+thorax”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The presence of D. immitis DNA in the head+thorax of mosquitoes indicates the presence of L3 infective stage.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In Iran, infection rates for An. maculipennis were 11.7% (4/34) and for Cx. theileri were 10% (15/149).

    “much higher infection rates for An. maculipennis, 11.7% (4/34) and Cx. theileri, 10% (15/149) were found in Iran [21]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] Azari-Hamidian S, Yaghoobi-Ershadi MR, Javadian E, Abai MR, Mobedi I, Linton YM, et al. Distribution and ecology of mosquitoes in a focus of in northwestern Iran, with the first finding of filarial larvae in naturally infected local mosquitoes. Med Vet Entomol. 2009;23:111–21. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In other Southern European countries such as Italy, D. repens has been found in Cx. pipiens at much lower rates than D. immitis.

    “in contrast with recent findings of D. repens in other Southern European countries such as Italy, albeit in much lower rates than D. immitis, in Cx. pipiens [16,17]”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [16] Cancrini G, Magi M, Gabrielli S, Arispici M, Tolari F, Dell’Omodarme M, et al. Natural vectors of dirofilariasis in rural and urban areas of the Tuscan region, central Italy. J Med Entomol. 2006;43:574–9. source

      Not checked in the free run.

    • cites [17] Latrofa MS, Montarsi F, Ciocchetta S, Annoscia G, Dantas-Torres F, Ravagnan S, et al. Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis. Parasites Vectors. 2012;5:76. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Aedes detritus s.l. were not differentiated to species level because the available technique would preclude screening for dirofilarial DNA.

    “Aedes detritus s.l. were not differentiated as the technique available at the time of this study would preclude the screening for dirofilarial DNA.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ae. caspius had been found positive for D. immitis in the whole mosquito.

    “Aedes caspius had been found positive for the whole mosquito [17]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Latrofa MS, Montarsi F, Ciocchetta S, Annoscia G, Dantas-Torres F, Ravagnan S, et al. Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis. Parasites Vectors. 2012;5:76. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Cx. pipiens infection rates in Spain, Italy, and Turkey have been reported as 0.3%, 0.048-0.54%, and 0.12%, respectively.

    “Infection of Cx. pipiens in Spain has been detected with a rate of 0.3% [15], in Italy it ranges from 0.048% [17], much lower than this present study, to 0.26% and 0.54% [16,44], similar to our values, with an intermediate prevalence rate in Turkey of 0.12% [18].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [15] Morchón R, Bargues MD, Latorre JM, Melero-Alcíbar R, Pou-Barreto C, Mas-Coma S, et al. Haplotype H1 of Culex pipiens implicated as a natural vector of Dirofilaria immitis in an endemic area of western Spain. Vector Borne Zoon Dis. 2007;7:653–8. source

      Not checked in the free run.

    • cites [17] Latrofa MS, Montarsi F, Ciocchetta S, Annoscia G, Dantas-Torres F, Ravagnan S, et al. Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis. Parasites Vectors. 2012;5:76. source

      Not checked in the free run.

    • cites [16] Cancrini G, Magi M, Gabrielli S, Arispici M, Tolari F, Dell’Omodarme M, et al. Natural vectors of dirofilariasis in rural and urban areas of the Tuscan region, central Italy. J Med Entomol. 2006;43:574–9. source

      Not checked in the free run.

    • cites [44] Cancrini G, Scaramozzino P, Gabrielli S, di Paolo M, Toma L, Romi R. Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in central Italy. J Med Entomol. 2007;44:1064–6. source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    Infection rates for An. maculipennis s.l. and Ae. caspius should be interpreted with caution.

    “therefore, infection rates should be interpreted with caution”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In Portugal, temperature increases have reached 0.5°C/decade since 1970, more than twice the global median temperature.

    “temperature increases have reached 0.5°C/decade since 1970, more than twice higher than the global median temperature [54]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [54] Ramos AM, Trigo RM, Santo FE. Evolution of extreme temperatures over Portugal: recent changes and future scenarios. Clim Res. 2011;48:177–92. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In continental Portugal, there are 41 identified species of mosquitoes.

    “In continental Portugal there are 41 identified species of mosquitoes [46]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [46] Ribeiro H, Ramos HC, Pires CA, Capela RA. An annotated checklist of the mosquitoes of continental Portugal (Diptera, Culicidae) . Actas do III Congresso Ibérico de Entomologia [Proceedings of the III Iberian Congress of Entomology], 1988, 233–254.

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Study design and conductnone given

    A 7-day period at 25°C may not be enough for the completion of the extrinsic cycle of D. immitis.

    “Although a 7 day period at 25°C may not be enough for the completion of the extrinsic cycle”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Future scenarios may range between 3-5.8°C by 2040-2090.

    “with future scenarios that may range between 3–5.8°C by 2040–2090”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [55] Casimiro E, Calheiros J, Santos FD, Kovats S. National assessment of human health effects of climate change in Portugal: approach and key findings. Environ Health Perspect. 2006;114:1950–6. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    This finding is in agreement with previous surveys for Cx. pipiens s.l. by both methods.

    “in agreement with previous surveys, namely for Cx. pipiens s.l., by both methods, as a sign of identical capture yield for this species [26]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Almeida APG, Galão RP, Sousa CA, Novo MT, Parreira R, Pinto J, et al. Potential mosquito vectors of arboviruses in Portugal: species, distribution, abundance and West Nile infection. Transact Royal Soc Trop Med Hygiene. 2008;102:823–32. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In Madeira, Cx. theileri has been found at infection rates of circa 0.9%-1.13%.

    “and circa 0.9%-1.13% in Madeira [19]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Santa-Ana M, Khadem M, Capela R. Natural infection of Culex theileri (Diptera, Culicidae) with Dirofilaria immitis (Nematoda, Filarioidea) on Madeira Island, Portugal. J Med Entomol. 2006;43:104–6. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    A compromise had to be taken to compensate for mosquito mortality and filarial DNA degradation while allowing for complete digestion of bloodmeals.

    “a compromise had to be taken to compensate for mosquito mortality and filarial DNA degradation, while allowing for complete digestion of bloodmeals.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Dirofilariasis is recognized as an expanding zoonosis, particularly in Europe.

    “dirofilariasis is recognized as an expanding zoonosis, particularly in Europe [4,5]”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Traversa D, Cesare A, Conboy G. Canine and feline cardiopulmonary parasitic nematodes in Europe: emerging and underestimated. Parasites Vectors. 2010;3:62. source

      Not checked in the free run.

    • cites [5] Simón F, Siles-Lucas M, Morchón R, González-Miguel J, Mellado I, Carretón E, et al. Human and animal dirofilariasis: the emergence of a zoonotic mosaic. ClinMicrobiol Rev. 2012;25:507–44.

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In the Canaries, Cx. theileri has been found at an infection rate of 0.16%.

    “Cx. theileri has been found at infection rates of 0.16% in the Canaries [20]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] Morchón R, Bargues MD, Latorre-Estivalis JM, Pou-Barreto C, Melero-Alcibar R, Moreno M, et al. Molecular Characterization of Culex theileri from Canary Islands, Spain, a potential vector of Dirofilaria immitis. J Clinic Experiment Pathol. 2011;S3:001. doi:10.4172/2161-0681.S3-001.

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    There is not enough data to draw conclusions on the seasonal dynamics of mosquitoes because there were only three collecting moments per year.

    “considering there were only three collecting moments per year, there is not enough data to draw conclusions on the seasonal dynamics of mosquitoes.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Ae. caspius, An. maculipennis s.l., An. atroparvus, Cx. pipiens of both bioforms pipiens and molestus, and Ae. detritus s.l. can support the development of D. immitis.

    “but also Ae. caspius, An. maculipennis s.l., An. atroparvus, Cx. pipiens of both bioforms pipiens and molestus and Ae. detritus s.l. can support the development of D. immitis”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Cx. quinquefasciatus has not been recorded in Portugal.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IR tends to be a more adequate method to capture An. maculipennis s.l.

    “as IR tends to be a more adequate method to capture this species [51]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [51] Lourenço PM, Sousa CA, Seixas J, Lopes P, Novo MT, Almeida AP. Anopheles atroparvus density modeling using MODIS NDVI in a former malarious area in Portugal. J Vector Ecol. 2011;36:279–91. source

      Not checked in the free run.

48 of 48

  • The authors state this as an assumption.

    A mosquito that takes a blood meal on a microfilaremic host on a day when DDU30 ≥130 can complete the extrinsic cycle and become infective, assuming an average mosquito lifespan of 30 days, regardless of temperatures below 14°C during that period.

    “When DDU30 is ≥130, it is assumed that a mosquito that might have taken a blood meal on a microfilaremic host on that particular day, had the possibility of allowing the completion of the extrinsic cycle, hence becoming infective, admitting an average mosquito life span of 30 days [2,13,40], independently of temperatures lower than 14°C during that period [41].”

  • The paper relies on this without support.

    The presence of D. immitis DNA in the head+thorax of mosquitoes indicates the presence of L3 infective stage.

    “based on the presence of filarial DNA in the head+thorax”

  • The paper relies on this without support.

    Cx. quinquefasciatus has not been recorded in Portugal.

    “although it has not yet been recorded in Portugal”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Table 3 reports zero positive pools in its total row

    Minor

    Table 3 reports zero positive head+thorax pools, abdomen pools, and pools overall in its total row. Table 4 reports 23, 41, and 54, respectively, for the same 9,156 collected and 5,866 tested mosquitoes; those values also match the positive rows in Table 3.

  • Culex pipiens tests are described as individual but tabulated as pools

    Minor

    The methods say Cx. pipiens s.l. specimens were analyzed individually. Table 3 lists 1,595 specimens tested in only 1,123 pools, so they cannot all have been tested individually as stated.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 6

  1. McCall JW, Genchi C, Kramer LH, Guerrero J, Venco L. Heartworm disease in animals and humans. AdvParasitol. 2008;66:193–285.
  2. Świątalska A, Demiaszkiewicz AW. First autochthonous case of Dirofilaria immitis invasion in dog in Poland. ŽycieWeterynaryjne. 2012;87:685.
  3. Ribeiro H, Ramos HC, Pires CA. Contribuição para o estudo dos vectores de filaríases animais em Portugal. J Socied Ciências Médicas. 1983;147:143–6.
  4. Ribeiro H, Ramos HC. Identification keys of the mosquitoes of continental Portugal, Azores and Madeira. Eur Mosquito Bull. 1999;3:1–11.
  5. BLAST® Basic Local Alignment Search Tool. http://blast.ncbi.nlm.nih.gov/Blast.cgi.
  6. Ribeiro H, Ramos HC, Pires CA, Capela RA. An annotated checklist of the mosquitoes of continental Portugal (Diptera, Culicidae) . Actas do III Congresso Ibérico de Entomologia [Proceedings of the III Iberian Congress of Entomology], 1988, 233–254.

Cited by

Sorted by citations

Related

Authors
Ferreira, C. A. C., de Pinho Mixão, V., Novo, M. T. L. M., Calado, M. M. P., Gonçalves, L. A. P., Belo, S. M. D., & de Almeida, A. P. G.
Affiliations
  • Universidade Nova de Lisboa (PT)
  • University of Lisbon (PT)
ORCID
Venue
Parasites & Vectors
Publisher
Springer Science and Business Media LLC
Published
2015-03-03
Volume
8
Issue
1
Pages
139-139
ISSN
1756-3305 (ISSN-L)
DOI
10.1186/s13071-015-0760-2
PMC
PMC4369802
OpenAlex
W2149798159
Cited by
61
References
51
Citations by year
2026: 4 · 2025: 5 · 2024: 5 · 2023: 9 · 2022: 4 · 2021: 7 · 2020: 7 · 2019: 4 · 2018: 3 · 2017: 3 · 2016: 7 · 2015: 3
FWCI
4.14
Type
journal article
Language
en
Open access
Licence
Locations
  • Parasites & Vectors (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • UpSpace Institutional Repository (University of Pretoria) (submittedVersion, other-oa)
  • Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, other-oa)
Funders
  • Fundação para a Ciência e a Tecnologia (PTDC/SAU-SAP/113523/2009)
  • Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa
Topics
  • Health Sciences › Medicine › Infectious Diseases › Parasitic Diseases Research and Treatment
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Research on Leishmaniasis Studies
  • Health Sciences › Veterinary › Small Animals › Helminth infection and control
Keywords
Dirofilaria immitis, canine dirofilariasis, mosquito vectors, Culex theileri, Culex pipiens, Anopheles maculipennis, Aedes caspius, Aedes detritus, Anopheles atroparvus, Dirofilaria repens, mosquito abundance, continental Portugal, filarial transmission
MeSH terms
Aedes, Animals, Anopheles, Culex, Dirofilaria immitis, Dirofilariasis, Dogs, Female, Insect Vectors, Larva, Culicidae, Portugal, Seasons, Polymerase Chain Reaction, DNA, Helminth

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

Statistics and arithmetic are recomputed, not judged.

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