Morphological and molecular differentiation between Culicoides oxystoma and Culicoides kingi (Diptera: Ceratopogonidae) in Tunisia

Background Culicoides kingi and Culicoides oxystoma belong to the Schultzei group of biting midges. These two species are vectors of disease in livestock of economic importance. As described in the literature, morphological identification for discrimination between them is still unclear. However, species-specific identification is necessary to solve taxonomic challenges between species and to understand their roles in disease transmission and epidemiology. This study aims to develop accurate tools to discriminate C. oxystoma from C. kingi using traditional morphometry and polymerase chain reaction-restriction fragment length polymorphism (PCR RFLP) assays for use in developing countries. Methods Specimens were collected from the region of Kairouan in central Tunisia. A total of 446 C. oxystoma/C. kingi individuals were identified using traditional morphometric analyses combined with PCR–RFLP of the cytochrome c oxidase subunit I gene. Thirteen morphometric measurements were performed from the head, wings, and abdomen of slide-mounted specimens, and six ratios were calculated between these measurements. Multivariate analyses of the morphometric measurements were explored to identify which variables could lead to accurate species identification. Results Four variables, namely antennae, wings, spermathecae, and palpus length, were suitable morphometric characteristics to differentiate between the species. Digestion with the SspI restriction enzyme of the PCR product led to good discriminative ability. Molecular procedures and phylogenetic analysis confirmed the efficiency of this simple and rapid PCR–RFLP method. Conclusions This study highlights for the first time in Tunisia the presence of C. oxystoma and its discrimination from C. kingi using abdominal measurements and the PCR–RFLP method. This approach could be applied in future epidemiological studies at the national and international levels. Graphical Abstract

Two Rare Cases of Bi-Abdomen and a Polar Gynandromorphismin ‎Culicoides (Biting Midges) from Makurdi, Nigeria

Three developmental abnormalities were found out of a total sample of eight hundred and twenty two within the genus Culicoides. These are two rare cases of double abdomen in females of the Schultzei and Imicola groups and a polar gynandromorphism, all in the genus Culicoides (biting midges) from Makurdi, Benue State, Nigeria. These females show double symmetrical abdomens equally attached to the meta-thorax and are related both on dorsal and ventral relative positions as well as larger and smaller relative capacities. The bi-abdomens in the Culicoides oxystoma (Schultzei group) were also separated by a speculated third rudimentary abdomen similarly attached to the meta-thorax; both abdomens possess three spermathecae (receptacula seminis) each but two spermathecae in the Culicoides imicola (Imicola group). The dorsal abdomen possesses eleven spiracles while the ventral sac, ten in the Culicoides oxystoma and revealed evidence of functionality; the dorsal abdomen was observed filled while the ventral only shows a smaller content volume. Both bi-abdominal specimens demonstrated no other apparent morphological dysfunction of other parts. On the other hand, the polar gynandromorphic specimen on stereomicroscopy revealed a typical female anterior portion and a posterior portion characteristic of male adult Culicoides. Further examination of the specimen showed absence of spermatheca. These abnormalities are the first reports of any type in invertebrates in Nigeria.

Effect of Environment and Elevation on Seasonal Prevalence of Culicoides in West Bengal

Background: Culicoides, popularly known as ‘biting midges’ is a group of vector insects causing transmission of pathogens to livestock and other closely related mammals leading to animal health deterioration. They are among the smallest and most abundant hematophagous vectors of Blue tongue virus (BTV), Epizootic haemorrhagic disease virus (EHDV), African horse sickness virus (AHSV), Equine encephalitis virus (EEV), protozoans, filarial nematodes throughout the world. They are known to spread livestock diseases in India in the states of Andhra Pradesh, Assam, Gujarat, Haryana, Himachal Pradesh, Jammu and Kashmir, Jharkhand, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Nagaland, Punjab, Rajasthan, Sikkim, Tamil Nadu, Tripura, Uttar Pradesh and West Bengal. 30 Culicoides vectors are reported worldwide, 13 of them from India, of which 11 are found in West Bengal. In the Indian subcontinent, biting midges live throughout the year, with two distinct peaks - one in the monsoon (August-September) and another peak during post-monsoon or beginning of winter (November) and there is a steady decrease during the summer months (May). Adults thrive best at low-temperature range (19-32oC) and rainfall plays an important role in Culicoides breeding and life-cycle. But a detailed account on the ecology, breeding sites, biology and bionomics of immature and adults have been poorly understood with very scanty literature. The present research is aimed to provide an exhaustive detail on the ecological behavior of Culicoides and their prevalence pattern based on the effect of meteorological parameter and elevation across different regions of West Bengal. Methods: Insect samples were collected using sweep nets in the early morning, afternoon and just before sunset for a period of four years from January 2014 to December 2017 in six agro-climatic regions of West Bengal. After collection, they were transferred in 70% ethanol, kept in a cool place, sorted and different parts of the specimen were mounted on glass slides in the phenol-balsam mixture and identified through Leica DFC 295 binocular light microscope. Ecological graph analysis were generated using software PAST 3.0 and MS-EXCEL 2016. Maps on sampling sites was generated using software ARC GIS 10.5). Result: Five Culicoides species-Culicoides oxystoma, C. actoni, C. peregrinus,C. orientalis and C. anophelis were collected. The results show highest prevalence in post-monsoon months at the coastal-saline region, at an of elevation 5-15m with ideal temperature of 14-35oC and rainfall of 1100-2000mm in West Bengal, with the highest dominance of Culicoides oxystoma throughout the year.

Culicoides Oxystoma a Potential Vector for Transmission of Bluetongue Virus 16 in Southern India

Bluetongue (BT) is a Culicoides vector borne disease of domestic and wild ruminants. It is caused by bluetongue virus (BTV). BT is infectious but non-contagious disease. The head–thorax region of Culicoides Oxystoma vector trapped on animal farm in TN was triturated and inoculated in BHK- 21 cell culture. After few blind passages it showed cytopathic effect (CPE) in cell culture. After appearance of 75% cytopathic effect in BHK 21 cell culture, viral nucleic acid was extracted. The RNA-PAGE analysis showed BTV specific characteristics migration pattern of 3:3:3:1. Viral nucleic acid was allowed for cDNA synthesis followed by NS1 and VP2 gene based RT-PCR. The NS1 gene RT-PCR, CPE and specific migration pattern in RNA-PAGE confirmed the sample as BTV. The VP2 gene based serotype specific RT-PCR identified the isolate as BTV serotype 16. The study suggested that Culicoides oxystoma could be a potential vector for transmission of BTV in southern India.