A hidden deadly venomous insect: First eco-epidemiological assessment and risk mapping of lonomism in Argentina.

BACKGROUND: Envenomation by the South American Lonomia saturniid caterpillars, named lonomism, constitutes an emerging and somewhat neglected public health issue in Argentina and neighboring countries. Considering that there is an intricate relationship between environment and human health in such cases, this study aimed to analyze the eco-epidemiological profile of 40 accidents and 33 occurrences of Lonomia spp. in Misiones (Argentina) between January 2014 and May 2020. METHODOLOGY/PRINCIPAL FINDINGS: We described the eco-epidemiological variables and characterized the abiotic scenario of such cases. Additionally, we obtained a density map that shows the punctual intensity of Lonomia records throughout Misiones. Most of the accidents occurred in the Department of Guaraní and involved male victims younger than 20 years old. The accidental/occasional occurrence of Lonomia spp. (considering both adult and caterpillar stages together) was significantly higher in the rural area, whereas only adult specimens were found in urban areas. We determined that the presence of this insect in Misiones is positively related to higher temperatures and solar radiation, and larger precipitation and evapotranspiration throughout the year. CONCLUSION/SIGNIFICANCE: This study represents an initial step towards the global understanding of lonomism as a public health problem in Argentina. It provides a map of the risk level for this envenomation in Misiones, which could help authorities address public health policy efforts to implement sustainable strategies for prevention and response to this threat in Northeastern Argentina and neighboring regions.

Occurrence of Anisakis pegreffii (Nematoda: Anisakidae) Larvae in Imported John Dory (Zeus faber) from Senegalese Coast Sold in Turkish Supermarkets.

BACKGROUND: West African goatfish Pseudupeneus prayensis, bluespotted seabream Pagrus caeruleostictus and John Dory Zeus faber are commercially marketed as fresh and frequently imported from Senegalese coast (FAO area 34.3.12) in Turkish supermarkets. PURPOSE: The aim of the current study was to collect data of occurrence and molecular identification of Anisakis species in imported P. prayensis, P. caeruleostictus and Z. faber caught in the Senegalese coast and to support epidemiological report for a risk evaluation of Anisakis species in Turkish supermarkets. METHODS: Forty imported fish from each species at a total of 120 samples were investigated for the presence of Anisakis larvae. Based on ITS region of RFLP analysis Anisakis larvae were identified and randomly selected five larvae were also sequenced for further confirmation for cox2 gene. RESULTS: No Anisakis larvae were isolated from P. prayensis, P. caeruleostictus whereas Anisakis larvae were only found in Z. faber. A total of 156 Anisakis larvae were collected from Z. faber. All larvae were molecularly identified as Anisakis pegreffii. The prevalence (%), intensity and abundance of Anisakis infection in Z. faber were detected to be 82.5%, 8.3 and 6.8, respectively. CONCLUSION: This is the first assessment of the occurrence of A. pegreffii in imported Z. faber from the Senegalese coast in Turkish supermarkets. Moreover, consuming imported P. prayensis and P. caeruleostictus present low to non-existent risk for anisakiasis in Turkish consumers. Furthermore, the presence of A. pegreffii larvae in imported Z. faber from the Senegal waters could have public health implications in Turkish consumers.

Contribution to the Biodiversity Assessment of Mosquitoes (Diptera: Culicidae) in the Atlantic Forest in Santa Catarina, Brazil.

The mosquito fauna of a coastal area of Santa Catarina state, Brazil (Baixada do Maciambu), was assessed, and possible mosquito larval habitats were mapped. Five new species records for the state are presented, two of which also are new genera records. From the 24 recognized species present in the area, 28% were from the subfamily Anophelinae and 72% from the Culicinae. The community structure throughout a year, the relevance of the new findings, and the medical importance of some species are discussed.

Semi-quantitative differentiation of cyathostomin larval cultures by reverse line blot.

Cyathostomins are the most prevalent horse nematodes worldwide and over 50 species are described. The eggs and the infective larvae (L3) can easily be obtained or cultured from infected horses, but cannot be differentiated morphologically at species level. A reverse line blot (RLB) method based on the hybridization of a PCR fragment with a species specific probe, has previously been developed for the differentiation of individual eggs and/or L3s, but is too labor intensive for large scale studies. In the present study a RLB method on multiple pooled L3s for the semi-quantitative differentiation of cyathostomin larval cultures was developed and validated. First, the probability of the presence of a certain species within a pool was calculated as function of the frequency and the number of L3s within a pool. Ten L3s per pool were found to be optimal. Next, the probability, the chance of occurrence was calculated when 4 pools per culture were used. The probability distributions for 0, 1, 2, 3 or 4 positive pools were transformed into the corresponding median frequency of the cumulative probability: 0.014, 0.04, 0.08, 0.16 and 0.59, respectively. Based on these calculated probabilities, RLB on 10 L3s per pool and 4 pools per sample was validated by estimating the cross-hybridization, precision and accuracy in 3 groups of horses. First, absence of cross-hybridization was confirmed by differentiation of the same L3s (160 L3s from the 4 horses from group 1) in the RLB on individual as well as on pooled L3s. Cross-hybridization was excluded for 9 of the most common cyathostomins. Next, the precision and accuracy were determined by the differentiation of 10 replicates of 3 cultures from 3 horses from group 2 (1200 L3s). The coefficient of variation (CV) was between 0 and 0.90 and the accuracy was between 0.42 and 1.73. A Monte Carlo simulation based on the observed scores and associated probability distributions gave similar results as the use of a fixed median frequency. The LPGs obtained from 276 larval culture counts from a larger cohort (23 horses, group 3) were not significantly different from the LPGs obtained from summation of the LPG per species found by RLB on pooled L3s. The RLB on pooled L3s was found therefore an useful semi-quantitative method for the differentiation of the most common cyathostomin L3, with a workload of approximately one tenth of that of the RLB on individual L3s.

Development of novel multiplex microsatellite polymerase chain reactions to enable high-throughput population genetic studies of Schistosoma haematobium.

BACKGROUND: Human urogenital schistosomiasis caused by Schistosoma haematobium is widely distributed across Africa and is increasingly targeted for control and regional elimination. The development of new high-throughput, cost-effective molecular tools and approaches are needed to monitor and evaluate the impact of control programs on the parasite populations. Microsatellite loci are genetic markers that can be used to investigate how parasite populations change over time and in relation to external influences such as control interventions. FINDINGS: Here, 18 existing S. haematobium microsatellite loci were optimised to enable simultaneous amplification across two novel multiplex microsatellite PCR's, each containing nine loci. Methods were developed for the cost effective and rapid processing and microsatellite analysis of S. haematobium larval stages stored on Whatman-FTA cards and proved robust on miracidia and cercariae collected from Zanzibar and Niger. CONCLUSION: The development of these novel and robust multiplex microsatellite assays, in combination with an improved protocol to elute gDNA from Whatman-FTA fixed schistosome larval stages, enables the high-throughput population genetic analysis of S. haematobium. The molecular resources and protocols described here advance the way researchers can perform multi locus-based population genetic analyses of S. haematobium as part of the evaluation and monitoring of schistosomiasis control programmes.

'Direct PCR' optimization yields a rapid, cost-effective, nondestructive and efficient method for obtaining DNA barcodes without DNA extraction.

Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction ('direct PCR') and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands.

Molecular phylogeny and host specificity of the larval Eustrongylides (Nematoda: Dioctophmidae) from freshwater fish in China.

The nematodes Eustrongylides spp. collected from different fish species in China were examined for their intra- and interspecific evolutionary variations using the molecular markers mitochondrial cytochrome oxidase c subunit 1 (COI) gene and internal transcribed spacer (ITS) rDNA regions. The phylogenetic analysis indicated that Eustrongylides species are divided into 3 well-supported clades. The ITS divergence between the clades suggested that clades 2 and 3 might represent the same species, whereas clade 1 represent another cryptic species. The host specificity of these nematodes was analyzed according to prevalence data, host range, and phylogenetic information. Clade 1 was found in 4 fish species, i.e., Odontobutis obscurus, Silurus asotus, Culter mongolicus, and Acanthogobius flavimanus, but was predominant in the 2 perciform species, O. obscurus and A. flavimanus. Clade 2 was found in 3 fish species, Monopterus albus, Channa argus, and Channa asiatica, but was predominant in M. albus, reported to feed primarily on oligochaetes, the first intermediate host of Eustrongylides sp. Clade 3 was found in 9 species, but its low prevalence suggests accidental infection in all species. Although the larval nematode presented low host specificity, it exhibited some host preference.

Ancestral state reconstruction for Dendroctonus bark beetles: evolution of a tree killer.

While most bark beetles attack only dead or weakened trees, many species in the genus Dendroctonus have the ability to kill healthy conifers through mass attack of the host tree, and can exhibit devastating outbreaks. Other species in this group are able to successfully colonize trees in small numbers without killing the host. We reconstruct the evolution of these ecological and life history traits, first classifying the extant Dendroctonus species by attack type (mass or few), outbreaks (yes or no), host genus (Pinus and others), location of attacks on the tree (bole, base, etc.), whether the host is killed (yes or no), and if the larvae are gregarious or have individual galleries (yes or no). We then estimated a molecular phylogeny for a data set of cytochrome oxidase I sequences sampled from nearly all Dendroctonus species, and used this phylogeny to reconstruct the ancestral state at various nodes on the tree, employing maximum parsimony, maximum likelihood, and Bayesian methods. Our reconstructions suggest that extant Dendroctonus species likely evolved from an ancestor that killed host pines through mass attack of the bole, had individual larvae, and exhibited outbreaks. The ability to colonize a host tree in small numbers (as well as gregarious larvae and attacks at the tree base) apparently evolved later, possibly as two separate events in different clades. It is likely that tree mortality and outbreaks have been continuing features of the interaction between conifers and Dendroctonus bark beetles.

PCR-based species identification of Agriotes larvae.

Click beetle larvae within the genus Agriotes (Coleoptera: Elateridae), commonly known as wireworms, are abundant ground-dwelling herbivores which can inflict considerable damage to field crops. In Central Europe up to 20 species, which differ in their distribution, ecology and pest status, occur in arable land. However, the identification of these larvae based on morphological characters is difficult or impossible. This hampers progress towards controlling these pests. Here, we present a polymerase chain reaction (PCR)-based approach to identify, for the first time, 17 Agriotes species typically found in Central Europe. Diagnostic sequence information was generated and submitted to GenBank, allowing the identification of these species via DNA barcoding. Moreover, multiplex PCR assays were developed to identify the nine most abundant species rapidly within a single-step reaction: Agriotes brevis, A. litigiosus, A. obscurus, A. rufipalpis, A. sordidus, A. sputator, A. ustulatus, A. lineatus and A. proximus. The latter two species remain molecularly indistinguishable, questioning their species status. The multiplex PCR assays proved to be highly specific against non-agrioted elaterid beetles and other non-target soil invertebrates. By testing the molecular identification system with over 900 field-collected larvae, our protocol proved to be a reliable, cheap and quick method to routinely identify Central European Agriotes species.

A recent survey of mosquito fauna in Guangdong Province,southern China, with a review of past records [corrected].

The southern province of Guangdong has long been subject to endemic mosquito-borne diseases. In recent years, this region of China has experienced rapid, extensive economic development involving environmental change, making much of the scant knowledge of its mosquito fauna obsolete. This paper reviews previous mosquito surveys, some of which may be too old to be of relevance to present-day conditions, and presents the results of a recent survey of adult and immature mosquitoes. The main vectors of mosquito-borne diseases endemic to the area, such as dengue and Japanese encephalitis virus, develop in container habitats. A three-year survey was carried out, between 2004 and 2006, of 4131 breeding containers in residential areas and in open, sparsely populated areas, of which approximately 50% were positive for the presence of mosquitoes, and 10 156 larvae and pupae were collected and identified. Twelve species were found in both residential and sparsely populated areas: Aedes albopictus (Skuse) (Diptera: Culicidae), Ae. lineatopennis (Ludlow), Ae. vexans (Meigen), Tanakaius togoi (Theobald), Culex barraudi Edwards, Cx dispectus Bram, Cx malayi (Leicester), Cx pallidothorax Theobald, Cx quinquefasciatus Say, Cx sitiens Wiedemann, Lutzia fuscanus Wiedemann and Tripteroides bambusa (Yamada). Armigeres subalbatus (Coquillett) was found only in containers in villages, whereas Ae. macfarlanei Edwards, Cx mimeticus Noé, Cx sinensis Theobald, Cx vegans Wiedemann, Cx wilfredi Colless and Mansonia uniformis (Theobald) were found only in non- or sparsely populated areas. In residential areas, the rank order of most common species, as measured by the proportion of containers colonized, was Ae. albopictus > Cx quinquefasciatus > Lu. fuscanus, whereas in sparsely populated areas the rank order was Cx quinquefasciatus > Ae. albopictus > Lu. fuscanus. Light traps in non- or sparsely populated areas caught 5995 adult mosquitoes of 25 species, some of which are not container breeders. The most common species were: Anopheles sinensis Wiedemann, An. maculatus Theobald, An. minimus Theobald, Ta. togoi, Cx bitaeniorhynchus Giles, Cx malayi, Cx quinquefasciatus, Cx sinensis Theobald, Cx sitiens, Cx tritaeniorhynchus Giles and Lu. fuscanus. It is noteworthy that nine species caught had not been previously recorded in Guangdong Province, highlighting the deficient knowledge of the current composition and distribution of the mosquito fauna of this part of China.

[Assessment of the preimaginal stages of the ticks collected from small mammals in Western and Northern Caucasus (Acari: Ixodidae)].

The exact identification of the preimaginal stages of the ticks collected from small mammals in Western and Northern Caucasus as a result of ten-year route investigations allowed us to obtain new data on the geographic ranges, altitude and biotopic preferences, and host-parasite relations of 11 species. A wide range of the joint occurrence (in the same locality, on the same host species, on the same host individual) is recorded for the first time for 10 species. The cohabitation of the each of 10 species with other 2-9 species has been found. The number of the cases of joint occurrence of different ixodid species on the same host individual is proportionate to the collection period in one locality. The cohabitation did not observed only for the species Ixodes ghilarovi, a high mountain ixodid species, which is for the first time recorded in 7 localities of Northern and Western Caucasus.

Assessment of cattle grub (Hypoderma spp.) prevalence in northeastern Italy: an immunoepidemiological survey on bulk milk samples using ELISA.

Hypodermosis is a myiasis of cattle mainly found in the Northern Hemisphere. In Italy it has been reported primarily in southern regions with a farm prevalence reaching a peak of 85%, while few data are available in the north of the country. This paper reports the results of a serological survey carried out on hypodermosis in Vicenza province (northeastern Italy). During January and February 1999, bulk milk samples were collected from 224 dairy farms and tested, using an hypodermosis ELISA kit. Epidemiological data, such as farm location, herd size, number of cows, grazing management, treatments against Hypoderma larvae, presence of imported animals, were gathered and statistically analysed in relation to the serological results. In 15 farms the presence of warbles was monitored by manual palpation and larvae collected for identification.Ninety-seven (43.3%) samples were detected as positive, whereas 12 (5.4%) and 115 (51.3%) were inconclusive and negative, respectively. Thirteen farms were positive upon parasitological examination and the larvae collected were identified as Hypoderma bovis.Seroprevalence was highly correlated with the practice of free grazing and slightly, but significantly, correlated with the number of cows on the farm. The mean antibody level of positive herds was significantly higher on farms without imported animals. On farms with warbled animals there was a high inverse correlation between antibody level and quantity of milk in the tanks and the number of lactating cows. The ELISA test on bulk milk samples has been shown, under field conditions, to be a useful, cost-effective and easy test for the diagnosis of bovine hypodermosis.