Sampling of Culicoides with nontraditional methods provides unusual species composition and new records for southern Spain.

BACKGROUND: Culicoides midges have been well-studied in Spain, particularly over the last 20 years, mainly because of their role as vectors of arboviral diseases that affect livestock. Most studies on Culicoides are conducted using suction light traps in farmed environments, but studies employing alternative trapping techniques or focusing on natural habitats are scarce. METHODS: In the present study, we analyze Culicoides captured in 2023 at 476 sites in western Andalusia (southern Spain) using carbon dioxide-baited Biogents (BG)-sentinel traps across different ecosystems. RESULTS: We collected 3,084 Culicoides midges (3060 females and 24 males) belonging to 23 species, including the new species Culicoides grandifovea sp. nov. and the first record of Culicoides pseudolangeroni for Europe. Both species were described with morphological and molecular methods and detailed data on spatial distribution was also recorded. The new species showed close phylogenetic relations with sequences from an unidentified Culicoides from Morocco (92.6% similarity) and with Culicoides kurensis. Culicoides imicola was the most abundant species (17.4%), followed by Culicoides grandifovea sp. nov. (14.6%) and Culicoides kurensis (11.9%). Interestingly, Culicoides montanus was the only species of the obsoletus and pulicaris species complexes captured, representing the first record of this species in southern Spain. A total of 53 valid Culicoides species have been reported in the area, with 48 already reported in literature records and 5 more added in the present study. Information on the flight period for the most common Culicoides species is also provided. CONCLUSIONS: To the best of our knowledge, our study represents the most comprehensive effort ever done on nonfarmland habitats using carbon-dioxide baited suction traps for collecting Culicoides. Our data suggests that using carbon dioxide traps offers a completely different perspective on Culicoides communities compared with routinely used light traps, including the discovery of previously unrecorded species.

Dinosaur bonebed amber from an original swamp forest soil.

Dinosaur bonebeds with amber content, yet scarce, offer a superior wealth and quality of data on ancient terrestrial ecosystems. However, the preserved palaeodiversity and/or taphonomic characteristics of these exceptional localities had hitherto limited their palaeobiological potential. Here, we describe the amber from the Lower Cretaceous dinosaur bonebed of Ariño (Teruel, Spain) using a multidisciplinary approach. Amber is found in both a root layer with amber strictly in situ and a litter layer mainly composed of aerial pieces unusually rich in bioinclusions, encompassing 11 insect orders, arachnids, and a few plant and vertebrate remains, including a feather. Additional palaeontological data-charophytes, palynomorphs, ostracods- are provided. Ariño arguably represents the most prolific and palaeobiologically diverse locality in which fossiliferous amber and a dinosaur bonebed have been found in association, and the only one known where the vast majority of the palaeontological assemblage suffered no or low-grade pre-burial transport. This has unlocked unprecedentedly complete and reliable palaeoecological data out of two complementary windows of preservation-the bonebed and the amber-from the same site.

Reinstatement of Athis miastagma (Dyar, 1925) (Lepidoptera: Castniidae) as a valid species inhabiting the Pacific slope of Central Mexico.

Mexico is the Mesoamerican country with the highest number of Castniidae species (12), most of which are found in the dry regions of the country. Among these, Athis hechtiae (Dyar, 1910) was considered a species widely distributed in Mexico after Athis miastagma (Dyar, 1925) was synonymized with A. hechtiae over two decades ago. A genetic and morphological study, as well as details of the natural history of various populations of these two taxa have led us to restore Athis miastagma as a valid species (stat. rest.), increasing the number of castniids recorded in Mexico to 13.

The rise of West Nile Virus in Southern and Southeastern Europe: A spatial-temporal analysis investigating the combined effects of climate, land use and economic changes.

West Nile Virus (WNV) has recently emerged as a major public health concern in Europe; its recent expansion also coincided with some remarkable socio-economic and environmental changes, including an economic crisis and some of the warmest temperatures on record. Here we empirically investigate the drivers of this phenomenon at a European wide scale by constructing and analyzing a unique spatial-temporal data-set, that includes data on climate, land-use, the economy, and government spending on environmental related sectors. Drivers and risk factors of WNV were identified by building a conceptual framework, and relationships were tested using a Generalized Additive Model (GAM), which could capture complex non-linear relationships and also account for spatial and temporal auto-correlation. Some of the key risk factors identified in our conceptual framework, such as a higher percentage of wetlands and arable land, climate factors (higher summer rainfall and higher summer temperatures) were positive predictors of WNV infections. Interestingly, winter temperatures of between 2 °C and 6 °C were among some of the strongest predictors of annual WNV infections; one possible explanation for this result is that successful overwintering of infected adult mosquitoes (likely Culex pipiens) is key to the intensity of outbreaks for a given year. Furthermore, lower surface water extent over the summer is also associated with more intense outbreaks, suggesting that drought, which is known to induce positive changes in WNV prevalence in mosquitoes, is also contributing to the upward trend in WNV cases in affected regions. Our indicators representing the economic crisis were also strong predictors of WNV infections, suggesting there is an association between austerity and cuts to key sectors, which could have benefited vector species and the virus during this crucial period. These results, taken in the context of recent winter warming due to climate change, and more frequent droughts, may offer an explanation of why the virus has become so prevalent in Europe.

The invasive longhorn beetle Xylotrechus chinensis, pest of mulberries, in Europe: Study on its local spread and efficacy of abamectin control.

The invasive wasp-mimicking Tiger Longicorn Beetle, Xylotrechus chinensis, a potentially lethal pest of mulberry trees (Moraceae: Morus sp.), was first reported in Europe in 2018, although its colonization and establishment were estimated to have occurred during the year 2012 or earlier. In Catalonia the infestation spread from four towns and 44.1 km2 in 2018 to 12 towns and 378.1 km2 in 2020; in the studied town of Barberà del Vallès, infested trees rose from 16.21% in February 2016 to 59.29% in December 2018. Human safety in public parks and avenues is a concern, as beetle infestation increases the risk of falling branches. The main objective of this study was to evaluate how the infestation progresses over time, with and without abamectin treatment, and provide insights into female egg-laying preferences. Such knowledge helps contribute to management efforts to reduce expansion of the range of beetle infestation. Our statistical analysis shows that females prefer laying eggs on larger trees, on the highest part of trunks and on the crown base (this being more preferred than the trunk), and they do so on warmer, SW orientations rather than those facing N, NW and E. Emergence holes and gallery slits predict the spreading of infestations to new trees. An abamectin treatment (trunk injection) carried out at the end of April significantly reduced the number of new infestation. However, for maximum insecticide efficiency, the best time for treating with abamectin would be from mid-July to mid-August, when newly hatched larvae begin feeding on the phloem.

Influence of socio-economic, demographic and climate factors on the regional distribution of dengue in the United States and Mexico.

BACKGROUND: This study examines the impact of climate, socio-economic and demographic factors on the incidence of dengue in regions of the United States and Mexico. We select factors shown to predict dengue at a local level and test whether the association can be generalized to the regional or state level. In addition, we assess how different indicators perform compared to per capita gross domestic product (GDP), an indicator that is commonly used to predict the future distribution of dengue. METHODS: A unique spatial-temporal dataset was created by collating information from a variety of data sources to perform empirical analyses at the regional level. Relevant regions for the analysis were selected based on their receptivity and vulnerability to dengue. A conceptual framework was elaborated to guide variable selection. The relationship between the incidence of dengue and the climate, socio-economic and demographic factors was modelled via a Generalized Additive Model (GAM), which also accounted for the spatial and temporal auto-correlation. RESULTS: The socio-economic indicator (representing household income, education of the labour force, life expectancy at birth, and housing overcrowding), as well as more extensive access to broadband are associated with a drop in the incidence of dengue; by contrast, population growth and inter-regional migration are associated with higher incidence, after taking climate into account. An ageing population is also a predictor of higher incidence, but the relationship is concave and flattens at high rates. The rate of active physicians is associated with higher incidence, most likely because of more accurate reporting. If focusing on Mexico only, results remain broadly similar, however, workforce education was a better predictor of a drop in the incidence of dengue than household income. CONCLUSIONS: Two lessons can be drawn from this study: first, while higher GDP is generally associated with a drop in the incidence of dengue, a more granular analysis reveals that the crucial factors are a rise in education (with fewer jobs in the primary sector) and better access to information or technological infrastructure. Secondly, factors that were shown to have an impact of dengue at the local level are also good predictors at the regional level. These indices may help us better understand factors responsible for the global distribution of dengue and also, given a warming climate, may help us to better predict vulnerable populations on a larger scale.

A New Alien Invasive Longhorn Beetle, Xylotrechus chinensis (Cerambycidae), Is Infesting Mulberries in Catalonia (Spain).

In this paper, the invasion of a new alien beetle species to Europe, the longhorn Xylotrechus chinensis (Chevrolat) (Cerambycidae), originating from East Asia, is revealed. It has settled in Catalonia (Spain), occupying at present an area of at least 44.1 km², where it has been shown to severely infest (ca. 10 to 45%) and eventually kill mulberry trees in private and public grounds. The main objective of this study was to evaluate its impact and provide new significant insights into its life history, seasonality, reproductive capacity (females produce an average of 83.4 ± 9.02 eggs) and the type of damage produced to mulberries. Such damage was thoroughly described to facilitate inspection by others. At least in laboratory conditions, X. chinensis has not used common grape vines as an alternative hostplant. Both plants, mulberries and grape vines, are important in Catalonia and Spain, the former providing shade and ornament to many streets and avenues, and the latter having great economic significance in Mediterranean wine production areas. Possible control methods to hinder its spread are suggested and one local wasp, Stephanus serrator (Stephanidae), was identified as a likely parasitoid. We believe the risk of this beetle widely spreading in Europe is very real.

Sexual communication in castniid moths: Males mark their territories and appear to bear all chemical burden.

Castniid moths (Lepidoptera: Castniidae) display a butterfly-like reproductive behavior, i.e., they use visual stimuli for mate location and females have apparently lost their pheromone glands in an evolutionary context. In this paper we report for the first time the identification of three new compounds, namely n-octadecyl acetate, (Z)-9-octadecenyl acetate and (E,Z)-2,13-octadecadienyl acetate, in males of the Castniid Palm Borer, Paysandisia archon, which could be involved in its short-range courtship behavior, and also shed light on recent controversies on the sexual behavior of the species. The compounds are produced in a ring-shaped gland of the male terminalia and have occasionally been detected in very minor amounts (ng) in ovipositor extracts of females, but only while mating or just after copulation. We also report that males use the already known (E,Z)-2,13-octadecadienol to mark their territory by rubbing their midlegs against the upper side of nearby leaves, especially palm leaves. This compound, produced in large amounts, is mostly concentrated in the midleg basitarsi and its maximum production is detected on the sexually mature 1-day-old specimens. In addition, analysis of male wings extracts confirms the presence of Z,E and E,E-farnesals, which are mostly produced in the median band of hindwings of 48-53 h-old insects. The biological significance of farnesals in this species is unknown. Our results point out that the chemical communication of P. archon relies mostly on males, which appear to bear all chemical burden in this respect.

Sexual communication in day-flying Lepidoptera with special reference to castniids or 'butterfly-moths'.

Butterflies and moths are subject to different evolutionary pressures that affect several aspects of their behaviour and physiology, particularly sexual communication. Butterflies are day-flying insects (excluding hedylids) whose partner-finding strategy is mainly based on visual cues and female butterflies having apparently lost the typical sex pheromone glands. Moths, in contrast, are mostly night-flyers and use female-released long-range pheromones for partner-finding. However, some moth families are exclusively day-flyers, and therefore subject to evolutionary pressures similar to those endured by butterflies. Among them, the Castniidae, also called 'butterfly-moths' or 'sun-moths', behave like butterflies and, thus, castniid females appear to have also lost their pheromone glands, an unparallel attribute in the world of moths. In this paper, we review the sexual communication strategy in day-flying Lepidoptera, mainly butterflies (superfamily Papilionoidea), Zygaenidae and Castniidae moths, and compare their mating behaviour with that of moth families of nocturnal habits, paying particular attention to the recently discovered butterfly-like partner-finding strategy of castniids and the fascinating facts and debates that led to its discovery.

Towards the PCR-based identification of Palaearctic Culicoides biting midges (Diptera: Ceratopogonidae): results from an international ring trial targeting four species of the subgenus Avaritia.

BACKGROUND: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses. To understand the role of Culicoides in the transmission of these viruses, it is essential to correctly identify the species involved. Within the western Palaearctic region, the main suspected vector species, C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, have similar wing patterns, which makes it difficult to separate and identify them correctly. METHODS: In this study, designed as an inter-laboratory ring trial with twelve partners from Europe and North Africa, we assess four PCR-based assays which are used routinely to differentiate the four species of Culicoides listed above. The assays based on mitochondrial or ribosomal DNA or microarray hybridisation were tested using aliquots of Culicoides DNA (extracted using commercial kits), crude lysates of ground specimens and whole Culicoides (265 individuals), and non-Culicoides Ceratopogonidae (13 individuals) collected from across Europe. RESULTS: A total of 800 molecular assays were implemented. The in-house assays functioned effectively, although specificity and sensitivity varied according to the molecular marker and DNA extraction method used. The Obsoletus group specificity was overall high (95-99%) while the sensitivity varied greatly (59.6-100%). DNA extraction methods impacted the sensitivity of the assays as well as the type of sample used as template for the DNA extraction. CONCLUSIONS: The results are discussed in terms of current use of species diagnostic assays and the future development of molecular tools for the rapid differentiation of cryptic Culicoides species.

Moths behaving like butterflies. Evolutionary loss of long range attractant pheromones in castniid moths: a Paysandisia archon model.

BACKGROUND: In the course of evolution butterflies and moths developed two different reproductive behaviors. Whereas butterflies rely on visual stimuli for mate location, moths use the 'female calling plus male seduction' system, in which females release long-range sex pheromones to attract conspecific males. There are few exceptions from this pattern but in all cases known female moths possess sex pheromone glands which apparently have been lost in female butterflies. In the day-flying moth family Castniidae ("butterfly-moths"), which includes some important crop pests, no pheromones have been found so far. METHODOLOGY/PRINCIPAL FINDINGS: Using a multidisciplinary approach we described the steps involved in the courtship of P. archon, showing that visual cues are the only ones used for mate location; showed that the morphology and fine structure of the antennae of this moth are strikingly similar to those of butterflies, with male sensilla apparently not suited to detect female-released long range pheromones; showed that its females lack pheromone-producing glands, and identified three compounds as putative male sex pheromone (MSP) components of P. archon, released from the proximal halves of male forewings and hindwings. CONCLUSIONS/SIGNIFICANCE: This study provides evidence for the first time in Lepidoptera that females of a moth do not produce any pheromone to attract males, and that mate location is achieved only visually by patrolling males, which may release a pheromone at short distance, putatively a mixture of Z,E-farnesal, E,E-farnesal, and (E,Z)-2,13-octadecadienol. The outlined behavior, long thought to be unique to butterflies, is likely to be widespread in Castniidae implying a novel, unparalleled butterfly-like reproductive behavior in moths. This will also have practical implications in applied entomology since it signifies that the monitoring/control of castniid pests should not be based on the use of female-produced pheromones, as it is usually done in many moths.

Field studies on Culicoides (Diptera: Ceratopogonidae) activity and response to deltamethrin applications to sheep in northeastern Spain.

An enclosure trapping experiment compared numbers and engorgement of Culicoides spp. taken from treated sheep (7.5% deltamethrin) to Culicoides from untreated sheep. Attack rates were low (0.2/min), but 58% of Culicoides obsoletus s.l. and 67% of Culicoides parroti Kieffer engorged on untreated sheep, and no engorgement occurred on treated sheep on 0 and 4 d posttreatment. A UV light trap in a livestock barn collected eight Culicoides spp. (510 individuals), dominated by C. obsoletus (Meigen) (68%), Culicoides imicola Kieffer (19%), Culicoides circumscriptus Kieffer (8%), and Culicoides alazanicus Dzhafarov (4%). A more powerful but nonattractive fan trap collected five species (121 individuals) dominated by C. obsoletus (48%), C. imicola (36%), C. alazanicus (8%), and C. circumscriptus (7%). Parity of C. obsoletus and C. imicola did not vary between the light and fan traps. Engorged Culicoides in the barn (33 C. obsoletus and three C. imicola) had fed on sheep or goats (precipitin test).

Variations in the mitochondrial cytochrome c oxidase subunit I gene indicate northward expanding populations of Culicoides imicola in Spain.

Culicoides imicola is the main vector for bluetongue (BT) and African horse sickness (AHS) viruses in the Mediterranean basin and in southern Europe. In this study, we analysed partial mitochondrial cytochrome c oxidase subunit I (COI) gene to characterize and confirm population expansion of Culicoides imicola across Spain. The data were analysed at two hierarchical levels to test the relationship between C. imicola haplotypes in Spain (n = 215 from 58 different locations) and worldwide (n = 277). We found nineteen different haplotypes within the Spanish population, including 11 new haplotypes. No matrilineal subdivision was found within the Spanish population, while western and eastern Mediterranean C. imicola populations were very structured. These findings were further supported by median networks and mismatch haplotype distributions. Median networks demonstrated that the haplotypes we observed in the western Mediterranean region were closely related with one another, creating a clear star-like phylogeny separated only by a single mutation from eastern haplotypes. The two, genetically distinct, sources of C. imicola in the Mediterranean basin, thus, were confirmed. This type of star-like population structure centred around the most frequent haplotype is best explained by rapid expansion. Furthermore, the proposed northern expansion was also supported by the statistically negative Tajima's D and Fu's Fs values, as well as predicted mismatch distributions of sudden and spatially expanding populations. Our results thus indicated that C. imicola population expansion was a rapid and recent phenomenon.

Factors affecting Culicoides species composition and abundance in avian nests.

Mechanisms affecting patterns of vector distribution among host individuals may influence the population and evolutionary dynamics of vectors, hosts and the parasites transmitted. We studied the role of different factors affecting the species composition and abundance of Culicoides found in nests of the blue tit (Cyanistes caeruleus). We identified 1531 females and 2 males of 7 different Culicoides species in nests, with C. simulator being the most abundant species, followed by C. kibunensis, C. festivipennis, C. segnis, C. truncorum, C. pictipennis and C. circumscriptus. We conducted a medicationxfumigation experiment randomly assigning bird's nests to different treatments, thereby generating groups of medicated and control pairs breeding in fumigated and control nests. Medicated pairs were injected with the anti-malarial drug Primaquine diluted in saline solution while control pairs were injected with saline solution. The fumigation treatment was carried out using insecticide solution or water for fumigated and control nests respectively. Brood size was the main factor associated with the abundance of biting midges probably because more nestlings may produce higher quantities of vector attractants. In addition, birds medicated against haemoparasites breeding in non-fumigated nests supported a higher abundance of C. festivipennis than the rest of the groups. Also, we found that the fumigation treatment reduced the abundance of engorged Culicoides in both medicated and control nests, thus indicating a reduction of feeding success produced by the insecticide. These results represent the first evidence for the role of different factors in affecting the Culicoides infracommunity in wild avian nests.

Biting rates of Culicoides midges (Diptera: Ceratopogonidae) on sheep in northeastern Spain in relation to midge capture using UV light and carbon dioxide-baited traps.

Biting midges in the genus Culicoides (Diptera: Ceratopogonidae) were collected near sunset by direct aspiration from sheep in northeastern Spain to determine species-specific biting rates and crepuscular activity. Midges were also collected by UV-baited light traps and CO2-baited traps over the same period to compare species diversity and abundance using these common surveillance methods to actual sheep attack rates. Culicoides aspirated from sheep included C. obsoletus, C. parroti, C. scoticus, C. punctatus, and C. imicola. Peak host-seeking activity during the time period examined for the two most commonly collected species (C. obsoletus and C. parroti) occurred just before sunset and activity ceased within 1 h after sunset. Host attack rates near sunset averaged 0.9 midges/min for both species with maximum attack rates of 3/min for C. obsoletus and 4/min for C. parroti. For both species, approximately 35% of midges collected from the sheep were engorged, giving a maximum biting rate of 1.1/min for C. obsoletus and 1.5/min for C. parroti. Traps baited with CO2 collected fewer midges of each species relative to other collection methods. Traps baited with UV light provided a good indication of species richness but significantly underestimated the host attack rate of C. obsoletus and C. parroti while overestimating the host attack rate of C. imicola. Animal-baited collecting is critical to interpret the epidemiological significance of light trap collections used for surveillance of the midge vectors of bluetongue virus and African horse sickness virus.

Mermithids (Nematoda: Mermithidae) of biting midges (Diptera: Ceratopogonidae): Heleidomermis cataloniensis n. sp. from Culicoides circumscriptus Kieffer in Spain and a species of Cretacimermis Poinar, 2001 from a ceratopogonid in Burmese amber.

Heleidomermis cataloniensis n. sp. (Nematoda: Mermithidae) is described from Culicoides circumscriptus Kieffer (Diptera: Ceratopogonidae) in Spain. Diagnostic characters include prominant elevations with multiple genital papillae on either side of the cloacal opening, only one row of genital papillae on the lateral surface of the tail, the tapering tip of the spicule and a reduced vagina. A male intersex of C. circumscriptus parasitised by H. cataloniensis n. sp. has mouthparts resembling those of the female. Two 100 million year-old fossil specimens of an un-named species of Cretacimermis Poinar, 2001, from an Early Cretaceous Burmese amber biting midge of the genus Leptoconops Skuse, show the antiquity of ceratopogonid-mermithid associations.

Differentiation of Culicoides obsoletus and Culicoides scoticus (Diptera: Ceratopogonidae) based on mitochondrial cytochrome oxidase subunit I.

Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle (Diptera: Ceratopogonidae) are sibling species of the Obsoletus group. This group comprises species of biting midges that are suspect vectors of bluetongue virus (family Reoviridae, genus Orbivirus, BTV) and African horse sickness virus (family Reoviridae, genus Orbivirus, AHSV). BTV and AHSV have been isolated several times from females of this group, although it has not been possible to determine the particular species harboring the virus, because of the inability to clearly identify the females of each species based on morphology. Both sexes of C. obsoletus and C. scoticus midges from Catalonia and the Balearics (Spain) were sequenced for the mitochondrial gene cytochrome oxidase subunit I (COI), and these sequences were analyzed to determine intra- and interspecific genetic variability. Species-specific primers for C. obsoletus and C. scoticus were designed and a polymerase chain reaction (PCR)-based diagnostic assay based on the COI gene and using a hemi-nested PCR technique was developed for reliably distinguishing the females of both species. The species-specific PCR diagnostic was compared with morphological discrimination of C. obsoletus and C. scoticus females. The morphologic characters were not fully reliable.

Results of current surveillance of likely bluetongue virus vectors of the genus Culicoides in Catalonia, Spain.

Following the outbreaks of bluetongue (BT) disease in sheep on the Balearic islands in 2000, a survey was conducted for Culicoides vectors along the eastern Catalonian cost of continental Spain where the presence of only C. obsoletus (Meigen) and C. pulicaris (Linnaeus) was known. Light-trap collections made at eight sites in 2002 yielded nine species of Culicoides, including C. imicola Kieffer (represented by a gravid female caught at Dosrius at a latitude of 41 degrees 35'N) and C. scoticus Downs and Kettle. The following season (2003), C. imicola was captured consistently at all sites and in greater numbers (maximum catch of 46) from August to November. The findings suggest that the distribution of C. imicola is extending northwards into Europe. The presence of four bluetongue vectors (C. imicola, C. obsoletus, C. pulicaris and C. scoticus) in Catalonia is of concern.