Diel activity and preferred landing sites in Culicoides biting midges attacking Fjord horses

In the summer of 2014, in the central part of The Netherlands, Culicoides spp. (Diptera: Ceratopogonidae) attack rates, biting rates, and preferred landing sites were determined for a pair of Fjord horses maintained permanently at pasture in an area devoid of cattle. Eleven body regions of the horses were screened for midges, each region sampled randomly for 5 min using a handheld mouth aspirator (pooter). Observations were confined to the hour immediately before and after sunset. Culicoides spp. were obtained from every body region, of which the four most abundant species – Culicoides chiopterus (Meigen), Culicoides punctatus (Meigen), the species complex Culicoides obsoletus (Meigen), and Culicoides dewulfi Goetghebuer – all were proven or potential vectors for arboviral diseases in livestock. Culicoides spp. activity was distinctly bimodal across the day, surging at sunset and 1 h after sunrise. Midges were inactive between 11:00 and 16:00 hours, these hours marking the time of day when horses can be pastured most safely but, thereafter, to avoid escalating attacks, would have to be stabled protectively. Around sunset, the mean attack rate of the four most abundant species ranged from 3.0 to 11.7 midges per min; of these, C. dewulfi and C. chiopterus were reared out of the dung of experimental horses. The Netherlands is home to the world's densest horse population (11 per km²), of which half are estimated to stay outdoors permanently with no access to protective housing. In the absence of a preventive vaccination policy, it is difficult to envisage how horses in northern Europe will be protected from infection during an outbreak of a Culicoides‐transmitted disease like African horse sickness.     

Emergence of Culicoides obsoletus group species from farm‐associated habitats in Germany

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) may transmit several arboviruses to ruminant livestock. The species of the Obsoletus group are considered to be among the most important vectors of bluetongue virus (BTV) in northern Europe. As agricultural environments offer suitable habitats for the development of their immature stages, the emergence of adult Culicoides from potential breeding sites was investigated at 20 cattle farms throughout Germany in 2012 and 2013. In analyses of species‐specific habitat preferences and relationships between Culicoides abundance in breeding substrates and their physicochemical characteristics, dungheaps emerged as the most important substrate for the development of Culicoides obsoletus sensu stricto (s.s.) (Meigen), whereas Culicoides chiopterus (Meigen) and Culicoides dewulfi Goetghebuer were generally restricted to cowpats. A decreasing pH value was associated with a higher abundance or a higher probability of observing these three species. Furthermore, the abundance of C. obsoletus s.s. was positively related to increasing moisture. Dungheaps were very productive breeding sites for this species and are therefore suggested as a target for potential control measures.     

Culicoides (Diptera: Ceratopogonidae) and livestock in the Netherlands: comparing host preference and attack rates on a Shetland pony,a dairy cow,and a sheep

Culicoides (Diptera: Ceratopogonidae) host preferences and attack rates were quantified in early summer at a dairy farm in the Netherlands using livestock tethered at pasture. Midges were aspirated hourly over seven consecutive hours (17:00–23:00) from a dairy cow, a Shetland pony, and a sheep and correspondingly yielded seventeen, thirteen, and nine species. Of the 14,181 midges obtained, approximately 95% belonged to the C. obsoletus complex, C. dewulfi, C. chiopterus, and C. punctatus that together include all proven or potential vectors for arboviral diseases in livestock in northwestern Europe. On average, 7.6 and 3.5 times more Culicoides were collected, respectively, from the cow and the Shetland pony than from the sheep. In descending order of abundance, the C. obsoletus complex, C. dewulfi, and C. chiopterus dominated attacks on all three hosts, whereas C. punctatus and C. pulicaris favored only the two larger hosts. Irrespective of the host species involved, the three body regions attracted the same component species, C. chiopterus favoring the legs, C. punctatus and C. achrayi the belly, and the C. obsoletus complex, C. dewulfi, and C. pulicaris the head, back, and flanks. That known and potential vectors for animal diseases feed indiscriminately on a broad range of mammal hosts means that all major livestock species, including equines, are rendered susceptible to one or more Culicoides‐borne pathogens.     

The Mondrian matrix: Culicoides biting midge abundance and seasonal incidence during the 2006–2008 epidemic of bluetongue in the Netherlands

During the northern Europe epidemic of bluetongue (BT), Onderstepoort‐type blacklight traps were used to capture Culicoides Latreille (Diptera: Ceratopogonidae) biting midges weekly between November 2006 and December 2008 on 21 livestock farms in the Netherlands. Proven and potential vectors for the bluetongue virus (BTV) comprised almost 80% of the midges collected: the Obsoletus complex, constituting C. obsoletus (Meigen) and C. scoticus Downes & Kettle (44.2%), C. dewulfi Goetghebuer (16.4%), C. chiopterus (Meigen) (16.3%) and C. pulicaris (Linnaeus) (0.1%). Half of the 24 commonest species of Culicoides captured completed only one (univoltine) or two (bivoltine) generations annually, whereas multivoltine species (including all BTV vectors) cycled through five to six generations (exceeding the one to four generations calculated in earlier decades). Whether this increment signals a change in the phenology of northern Europe Culicoides or simply is an adaptive response that manifests during warmer episodes, thus heightening periodically the incursive potential of midge‐borne arboviruses, remains to be clarified. Culicoides duddingstoni Kettle & Lawson, C. grisescens Edwards, C. maritimus Kieffer, C. pallidicornis Kieffer and C. riethi Kieffer are new records for the biting midge fauna of the Netherlands. It is suggested that C. punctatus (Meigen) be added to the European list of vector Culicoides.     

Host preferences of Palaearctic Culicoides biting midges: implications for transmission of orbiviruses

Feeding success depends on host availability, host defensive reactions and host preferences. Host choice is a critical determinant of the intensity at which pathogens are transmitted. The aim of the current study was to describe host preferences of Palaearctic Culicoides species (Diptera: Ceratopogonidae) Latreille using traps baited with the five different host species of poultry, horse, cattle, sheep and goat. Collections were carried out nightly in July and August 2009 in western France with three replicates of a 5 × 5 randomized Latin square (five sites, five hosts). Moreover, an ultraviolet (UV) light/suction trap was operated during host‐baited collections to correlate Culicoides biting rates and UV light/suction trap catches. A total of 660 Culicoides belonging to 12 species, but comprised mainly of Culicoides scoticus Downes and Kettle, Culicoides dewulfi Goetghebuer and Culicoides obsoletus Meigen, were collected on animal baits. Abundance was highest for the horse, which accounted for 95% of all Culicoides caught, representing 10 species. The horse, the largest bait, was the most attractive host, even when abundance data were corrected by weight, body surface or Kleiber's scaling factor. Culicoides obsoletus was the only dominant species attracted by birds. Both C. scoticus and C. dewulfi were collected mainly from the upper body of the horse. Finally, the quantification of host preferences allows for discussion of implications for the transmission of Culicoides‐borne pathogens such as bluetongue virus.     

Limited attractant range of the black‐light suction trap for the capture of Culicoides biting midges (Diptera: Ceratopogonidae)

The suction light trap (LT) is a standard tool used to capture Culicoides biting midges, when estimating abundances, and mapping species ranges. The exact range of attraction of the LT is in dispute, however, with several studies indicating the range to vary widely, between 4 and 50 m. In this study, the attractant range of a LT, set at various distances (0, 1, 2, 3, 5 and 8 m) from a cow tethered at pasture, is assessed in two trials conducted on a dairy farm in the Netherlands in 2013 and 2014. In the first trial, sweep‐netting and light‐trapping was performed concurrently. Although a Spearman Rank correlation of 0.89 (P = 0.03) showed the two sets of abundance results to be highly correlated, species prevalence differed significantly according to the method used: the Culicoides obsoletus complex dominated the LT observations, whereas Culicoides chiopterus and Culicoides dewulfi were more abundant in those obtained by sweep‐net, due to the former taxon being largely nocturnal, the latter diurnal. There is a distinct negative logarithmic relationship between Culicoides abundance and distance from the vertebrate host, with midge abundance, in the LT, increasing significantly only when it is placed very close to the bait animal, at a distance of 0–1 m. The sharp decay function in the number of Culicoides caught with increasing distance from the LT, limits the comparison of LT surveillance data obtained under various settings, even from within a climatically homogeneous geographical region such as north‐western Europe.     

Barcoding of biting midges in the genus Culicoides: a tool for species determination

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness virus and bluetongue virus. However, the identification of Culicoides based on morphological features is difficult. The sequencing of mitochondrial cytochrome oxidase subunit I (COI), referred to as DNA barcoding, has been proposed as a tool for rapid identification to species. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined Culicoides species in Swedish collections. In total, 237 specimens of Culicoides representing 37 morphologically distinct species were used. The barcoding generated 37 supported clusters, 31 of which were in agreement with the morphological determination. However, two pairs of closely related species could not be separated using the DNA barcode approach. Moreover, Culicoides obsoletus Meigen and Culicoides newsteadi Austen showed relatively deep intraspecific divergence (more than 10 times the average), which led to the creation of two cryptic species within each of C. obsoletus and C. newsteadi. The use of COI barcodes as a tool for the species identification of biting midges can differentiate 95% of species studied. Identification of some closely related species should employ a less conserved region, such as a ribosomal internal transcribed spacer.     

Phenotypic differentiation and phylogenetic signal of wing shape in western European biting midges,Culicoides spp., of the subgenus Avaritia

In the past decade biting midges of the subgenus Avaritia (Diptera: Ceratopogonidae) have been popular subjects of applied entomological studies in Europe owing to their implication as biological vectors in outbreaks of bluetongue and Schmallenberg viruses. This study uses a combination of cytochrome oxidase subunit I barcode sequencing and geometric morphometric analyses to investigate wing shape as a means to infer species identification within this subgenus. In addition the congruence of morphological data with different phylogenetic hypotheses is tested. Five different species of the subgenus Avaritia were considered in the study (C. obsoletus (Meigen); C. scoticus Kettle and Lawson; C. chiopterus (Meigen); C. dewulfi Goetghebuer and C. imicola (Kieffer)). The study demonstrated that over 90% of individuals could be separated correctly into species by their wing shape and that patterns of morphological differentiation derived from the geometric morphometric analyses were congruent with phylogenies generated from sequencing data. Morphological data produced are congruent with monophyly of the subgenus Avaritia and the exclusion of C. dewulfi from the group containing C. obsoletus, C. scoticus and C. chiopterus. The implications of these results and their importance in a wider context of integrating multiple data types to interpret both phylogeny and species characterization is discussed.     

First detection of endosymbiotic bacteria in biting midges Culicoides pulicaris and Culicoides punctatus,important Palaearctic vectors of bluetongue virus

Heritable bacteria have been highlighted as important components of vector biology, acting as required symbionts with an anabolic role, altering competence for disease transmission, and affecting patterns of gene flow by altering cross compatibility. In this paper, we tested eight U.K. species of Culicoides (Diptera: Ceratopogonidae) midge for the presence of five genera of endosymbiotic bacteria: Cardinium (Bacteroidales: Bacteroidaceae); Wolbachia (Rickettsiales: Rickettsiaceae); Spiroplasma (Entomoplasmatales: Spiroplasmataceae); Arsenophonus (Enterobacteriales: Enterobacteriaceae), and Rickettsia (Rickettsiales: Rickettsiaceae). Cardinium spp. were detected in both sexes of Culicoides pulicaris and Culicoides punctatus, two known vectors of bluetongue virus. Cardinium spp. were not detected in any other species, including the Culicoides obsoletus group, the main vector of bluetongue and Schmallenberg viruses in northern Europe. The other endosymbionts were not detected in any Culicoides species. The Cardinium strain detected in U.K. Culicoides species is very closely related to the Candidatus Cardinium hertigii group C, previously identified in Culicoides species in Asia. Further, we infer that the symbiont is not a sex ratio distorter and shows geographic variation in prevalence within a species. Despite its detection in several species of Culicoides that vector arboviruses worldwide, the absence of Cardinium in the C. obsoletus group suggests that infections of these symbionts may not be necessary to the arboviral vector competence of biting midges.     

Phylogenetic relationships of the subgenus AvaritiaFox, 1955 including Culicoides obsoletus (Diptera, Ceratopogonidae) in Italy based on internal transcribed spacer 2 ribosomal DNA sequences

Abstract. Biting midges of the genus Culicoides include vector species for orbiviral diseases, such as bluetongue and African horse sickness. Although the Afro-Asiatic species C. imicola is the major vector of bluetongue in the Mediterranean basin, recent outbreaks in regions where C. imicola is absent has incriminated other Culicoides, including those belonging to the Obsoletus Complex of the subgenus Avaritia Fox, 1955. The classical taxonomy of this species complex is unclear and this stimulated the molecular analysis of twenty Culicoides populations sampled from eighteen localities across Italy. Ribosomal internal transcribed spacer 2 sequences were used to characterize the intra- and interspecific variation between Italian members of the Obsoletus Complex and related species, by means of an analysis of molecular variance and phylogenetic analyses. Although morphological differentiation is often extremely difficult, the molecular analysis clearly demonstrated a high degree of divergence between most species. The study showed that at least seven species of the subgenus Avaritia occur in Italy; these are C. obsoletus, C. scoticus, C. montanus, C. dewulfi, C. imicola and two species that could not be identified with certainty, but one of which is similar to C. chiopterus. Finally, a simple polymerase chain reaction assay was developed that rapidly discriminates between four members of the Obsoletus Complex in Italy, a prerequisite for vector identification.     

Indoor and outdoor winter activity of Culicoides biting midges,vectors of bluetongue virus,in Italy

Indoor and outdoor winter activity of Culicoides spp. (Diptera: Ceratopogonidae) in central Italy was investigated in order to evaluate whether indoor activity might account for the overwintering of bluetongue virus, as has been hypothesized by some authors. Weekly Culicoides collections were performed at three farms over three consecutive winter seasons. At each farm, two black‐light traps were operated simultaneously, indoors and outdoors. Culicoides were identified using both morphological and molecular means. The Culicoides obsoletus group accounted for 98.2% of sampled specimens. Within this group, C. obsoletus s.s. accounted for 56.8% and Culicoides scoticus for 43.2% of samples. Nulliparous, parous and engorged females were caught throughout the entire winter, both indoors and outdoors. At times, indoor catch sizes outnumbered outdoor collections. A significant inverse correlation was found between minimum temperature and the proportion of indoor Culicoides of the total midge catch, thus indicating that lower outdoor temperatures drive Culicoides midges indoors. High rates of engorged females were recorded indoors, possibly as the result of the propensity of C. obsoletus females to feed indoors. Higher proportions of parous females were found in indoor than in outdoor catches, indicating higher survival rates indoors and, consequently, higher vectorial capacities of midges sheltering indoors compared with those remaining outdoors.     

Revealing potential bridge vectors for BTV and SBV: a study on Culicoides blood feeding preferences in natural ecosystems in Spain

Several species of Culicoides (Diptera: Ceratopogonidae) are vectors of pathogens, such as the bluetongue (BTV) and Schmallenberg (SBV) viruses, which cause important diseases in domestic and wild ruminants. As wild ruminants can contribute to overwintering and epizootics of both diseases, knowledge of the host‐feeding behaviour of Culicoides in natural ecosystems is important to better understand their epidemiology. Blood‐engorged Culicoides females trapped in natural areas inhabited by different wild ruminant species were genetically analysed to identify host species. The origin of bloodmeals was identified in 114 females of 14 species of Culicoides. A total of 104 (91.1%) Culicoides fed on mammals and 10 (8.9%) on birds. The most abundant host identified was red deer (66.7%), followed by humans (13%) and fallow deer (6.1%). Eleven of the 14 species of Culicoides fed exclusively on mammalian hosts. Among them, five are mammalophilic species considered to be important BTV and/or SBV vectors. The results of the present study confirm that Culicoides imicola, Culicoides obsoletus, Culicoides scoticus, Culicoides pulicaris and Culicoides punctatus fed on wild ruminants, and therefore support the hypothesis that these species can act as bridge vectors by facilitating the circulation of pathogens between wild and domestic ruminant communities.     

The dying of the light: crepuscular activity in Culicoides and impact on light trap efficacy at temperate latitudes

The light trap is the tool of choice for conducting large‐scale Culicoides (Diptera: Ceratopogonidae) vector surveillance programmes. Its efficacy is in doubt, however. To assess this, hourly changes in Culicoides activity over the 24‐h diel were determined comparatively by way of light trapping and aerial sweeping, and correlated against light intensity. In the Netherlands, sweeping around cattle at pasture revealed that, in early summer, Culicoides are active throughout the diel, and that their abundance peaks during the crepuscular period and falls to a low during the brightest hours of the day. By contrast, the light trap was able to accumulate Culicoides only at night (i.e. after illuminance levels had dropped to 0 lux and midge activity had begun to decline). Although Culicoides chiopterus and species of the Culicoides obsoletus complex were similarly abundant around livestock, they differed critically in their hours of peak activity, being largely diurnal and nocturnal, respectively. This polarity helps to explain why, routinely, the C. obsoletus complex dominates light trap collections and C. chiopterus does not. Inability to accumulate Culicoides at light intensity levels above 0 lux means that, at ever‐higher latitudes, particularly beyond 45° N, the progressive northward lengthening of the twilight period will have an increasingly adverse impact upon the efficacy of the light trap as a vector surveillance tool.     

The effect of ambient temperature fluctuations on Culicoides biting midges population dynamics and activity in dairy farms: a longitudinal study

The effect of climatic factors on the presence of Culicoides Latreille (Diptera: Ceratopogonidae) was previously studied. Nevertheless, lack of laboratory rearing data hampers species‐specific prediction of weather fluctuations effect on population size. To determine fluctuations in population size in the field, we recorded Culicoides and other Nematocerans in seven Israeli dairy farms over two‐years (2011–2012) and analysed the association of their dynamics with fluctuations in ambient temperature and total rainfall. In six farms, the most abundant species were Culicoides imicola Kieffer and Culicoides schultzei (Enderlein) gp., primarily composed of parous females, and in one farm Culicoides obsoletus (Meigen) gp., mostly nulliparous females, were dominant. While the total number of insects was similar in both years, Culicoides numbers were significantly higher in 2012, but appeared later in the season and reached a higher peak. A multi‐variable linear regression model demonstrated positive association of C. imicola and C. schultzei numbers with the monthly multi‐annual ambient temperature and its specific deviation, but not with monthly rainfall. C. obsoletus populations peaked at spring and sharply decreased when temperature exceeded 20 °C, and were best modelled by adding quadratic terms. Weather‐specific estimation of population size under field conditions may enable to predict outbreaks intensity of Culicoides‐borne viruses.     

Are Bogs Reservoirs for Emerging Disease Vectors? Evaluation of Culicoides Populations in the Hautes Fagnes Nature Reserve (Belgium)

Several species of Culicoides (Diptera: Ceratopogonidae) biting midges serve as biological vectors for the bluetongue virus (BTV) and the recently described Schmallenberg virus (SBV) in northern Europe. Since their recent emergence in this part of the continent, these diseases have caused considerable economic losses to the sheep and cattle industries. Much data is now available that describe the distribution, population dynamics, and feeding habits of these insects. However, little is known regarding the presence of Culicoides in unusual habitats such as peaty marshes, nor their potential vector capacity. This study evaluated Culicoides biting midges present in the bogs of a Belgian nature reserve compared to those residing at a nearby cattle farm. Culicoides were trapped in 2011 at four different sites (broadleaved and coniferous forested areas, open environments, and at a scientific station) located in the Hautes Fagnes Nature Reserve (Belgium). An additional light trap was operated on a nearby cattle farm. Very high numbers of biting midges were captured in the marshy area and most of them (70 to 95%) were Culicoides impunctatus, a potential vector of BTV and other pathogens. In addition, fewer numbers of C. obsoletus/C. scoticus species, C. chiopterus, and C. dewulfi were observed in the bogs compared to the farm. The wet environment and oligotrophic nature of the soil were probably responsible for these changes in the respective populations. A total of 297,808 Culicoides midges belonging to 27 species were identified during this study and 3 of these species (C. sphagnumensis, C. clintoni and C. comosioculatus) were described in Belgium for the first time.     

Laboratory and field evaluations of chemical and plant‐derived potential repellents against Culicoides biting midges in northern Spain

The efficacy of 23 compounds in repelling Culicoides biting midges (Diptera: Ceratopogonidae), particularly Culicoides obsoletus (Meigen) females, was determined by means of a Y‐tube olfactometer. The 10 most effective compounds were further evaluated in landing bioassays. The six most promising compounds (including chemical and plant‐derived repellents) were evaluated at 10% and 25% concentrations in field assays using Centers for Disease Control (CDC) light traps. At least three compounds showed promising results against Culicoides biting midges with the methodologies used. Whereas olfactometer assays indicated DEET at 1 µg/µL to be the most effective repellent, filter paper landing bioassays showed plant‐derived oils to be better. Light traps fitted with polyester mesh impregnated with a mixture of octanoic, decanoic and nonanoic fatty acids at 10% and 25% concentrations collected 2.2 and 3.6 times fewer midges than control traps and were as effective as DEET, which is presently considered the reference standard insect repellent. The best plant‐derived product was lemon eucalyptus oil. Although these have been reported as safe potential repellents, the present results indicate DEET and the mixture of organic fatty acids to be superior and longer lasting.     

Influence of carbon dioxide on numbers of Culicoides midges collected with suction light traps in South Africa

To implement risk management against diseases transmitted by species of Culicoides Latreille, 1809 (Diptera: Ceratopogonidae), it is essential to identify all potential vectors. Light traps are the most commonly used tool for the collection of Culicoides midges. Given the indiscriminate artificial attraction of light, traps will collect all night‐flying insects rather than only livestock‐associated Culicoides midges. Factors that may increase the efficacy of traps, especially for livestock‐associated Culicoides midges, require investigation. In the present study, results obtained with Centers for Disease Control (CDC) and Onderstepoort light traps baited with carbon dioxide (CO₂) were compared with those of unbaited controls. Comparisons were made using two replicates of a 4 × 4 randomized Latin square design. With both trap types, the mean numbers of Culicoides midges collected in 16 baited traps were higher than those caught in 16 unbaited traps. Although exceptionally low numbers were collected with the CDC traps, the increases in the numbers and frequency of collection of Culicoides imicola Kieffer, 1913 were more pronounced in the CDC traps compared with the Onderstepoort traps. These results indicate that the addition of CO₂ may increase the efficiency of these traps for the collection of C. imicola and other livestock‐associated Culicoides species.     

Bloodmeal analysis in Culicoides midges collected near horses,donkeys and zebras in the Eastern Cape,South Africa

An upsurge in African horse sickness (AHS) in the Eastern Cape, South Africa, from 2006 led to an epidemiological reassessment of the disease there. Light trapping surveys carried out near horses, donkeys and zebras in 2014–2016 collected 39 species of Culicoides midge (Diptera: Ceratopogonidae) that are potential vectors of AHS. To establish if these midges fed on equids, DNA sequences were obtained from the gut contents of 52 female midges (35 freshly blood‐fed, 13 gravid and four parous), representing 11 species collected across 11 sites. Culicoides leucostictus fed on all three equids. Culicoides bolitinos, Culicoides imicola and Culicoides magnus fed on both horses and donkeys. Culicoides onderstepoortensis fed on donkeys, and Culicoides similis and Culicoides pycnostictus fed on zebras. Bloodmeals from cows, pigs, warthogs, impalas and a domestic dog were also identified in various species, but none of the midges tested had fed on birds. These results contribute to knowledge of the vectorial capacity of several species of Culicoides with regard to AHS in the Eastern Cape and point to potential reservoir hosts, of which donkeys, zebras and domestic dogs have previously been found to harbour AHS. Blood‐fed midges were also obtained throughout winter, indicating the potential for endemic AHS in the province.     

Characterization of two cryptic species,Culicoides stigma and C.parroti (Diptera: Ceratopogonidae), based on barcode regions and morphology

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness, bluetongue, and Schmallenberg virus. The identification of Culicoides based on morphological features can be difficult. Three species of biting midges, Culicoides nubeculosus, C. stigma, and C. parroti have emerged in the laboratory from mud collected around watering troughs on a farm in northern France. Emerging Culicoides were characterized morphologically and molecularly using molecular markers. The closely related species C. stigma and C.parroti showed highly divergent sequences for both mitochondrial (cytochrome B and cytochrome oxidase I) and ribosomal DNA first internal transcribed spacer. A RFLP based on a single restriction using the same enzyme (HaeIII) for both cytochrome C oxidase I and cytochrome B is proposed to identify these species.