Host-seeking and exophilic-endophilic activities of Culicoides spp. (Diptera: Ceratopogonidae) with notes on two trapping methods

Abstract

Host-seeking behavior of Culicoides species was examined from 2018 to 2019 in West Bengal, India, which elucidated diel activity, feeding success, attack rate, biting rate, and preferential landing of adult Culicoides on the cattle. A comparative assessment was done between the light trap and the aspirator. The host-seeking experiment involved a substantial timeframe of 297 h of catch collections over 27 nights. The number of adult Culicoides captured in the light trap was 1.3 times higher than the aspirator collections. The species in light trap catch were Culicoides oxystoma Kieffer Culicoides peregrinus Kieffer, and Culicoides fulvus Sen and Das Gupta (Diptera: Ceratopogonidae). However, only C. oxystoma and C. peregrinus were collected using the aspirator. The findings related to feeding success, attack rate, and biting rate carried significant implications for the vectorial potential of C. oxystoma and C. peregrinus. The light trap data suggest that Culicoides species displayed crepuscular behavior, while the aspirator collections peaked 1 h before sunrise, between 04:00 and 05:00 h, and gradually declined. A separate study evaluated the exophily and endophily of Culicoides in 2016 at Memari in West Bengal. To delve into the indoor–outdoor activity, 264 trap collections were made in 4 combinations: Light trap operated in the presence and absence of cattle, placed outdoors and indoors. The study revealed that the outdoor prevalence of midges was 14 times higher than the indoor. There was a 6-time increase in the prevalence of adult Culicoides in the presence of cattle, indicating a preference for outdoor locations for feeding.

Graphical Abstract

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Introduction

The epidemiology of vectors of bluetongue virus (BTV) that causes bluetongue disease (BTD) hinges on understanding the host-seeking behavior of Culicoides (Diptera: Ceratopogonidae) (Allyòn et al. 2014). In India, the lack of understanding of the biology and ecology of the vector species presents a critical knowledge gap (Harsha et al. 2020). Further, Chanda et al. (2019) proposed BTV outbreak models based on landscape and climatic factors; however, they referred to the opinion of Purse et al. (2015) from the world domain to highlight the happenings related to the Culicoides life cycle, making it evident the lack of information in Indian Culicoides. The following species possibly pose a threat to the spread of BTV across India: Culicoides oxystoma Kieffer, Culicoides fulvus Sen and Das Gupta, Culicoides orientalis Macfie, Culicoides imicola Kieffer, Culicoides peregrinus Kieffer, and Culicoides brevitarsis Kieffer (Harrup et al. 2016). In this study, following species were investigated: Culicoides oxystoma, C. peregrinus, C. fulvus, Culicoides innoxius Sen and Das Gupta, and Culicoides anophelis Edwards. For instance, C. oxystoma was abundant near cattle (Harsha et al. 2020), sheep, and goats (Mazumdar et al. 2022) and has been identified to carry the BTV-1 serotype from Sardarkrushinagar and Jasdan in Gujarat (Dadawala et al. 2012). Blood meal analysis revealed that it feeds on humans, chickens, goats, pigs, and cattle (Li et al. 2024). Similarly, C. peregrinus has been observed in high numbers around livestock (Harsha et al. 2020), with BTV-23 being isolated from the species in Tamil Nadu (Ranjan et al. 2017). Although C. fulvus is known to occur near and around livestock (Harsha et al. 2020), the detection of any BTV serotype in this species in India is yet to be reported. Culicoides innoxius belongs to the same subgenus as C. peregrinus, i.e., Hoffmania, characterized by large size and dark pigmentation over the body (Wirth and Hubert 1989). Light traps operated in cattle sheds revealed the presence of blood-fed C. innoxius in significant quantities (Mazumdar and Mazumdar 2020). Li et al. (2024) reported the presence of a notable proportion of blood-engorged C. innoxius feeding on goats in their study from Yunnan province of China. Mazumdar et al. (2022) reported C. innoxius from a sheep pen in Jharkhand state of India. Culicoides anophelis is a species of concern due to its wide range of hosts ranging from livestock (Mazumdar et al. 2022) to mosquitoes (Ma et al. 2013) and its possible role in the transmission of Plasmodium and other pathogens of medical and veterinary importance (Laird 1947).

Presently, India recorded 535.8 million major farm animals, i.e., cattle, buffalo, poultry, sheep, and goats, including Indigenous and exotic breeds, with a bovine population of 302.8 million (buffalo, cattle, yak) (20th Livestock Census 2019). The diverse livestock populations increase the risk of BTV transmission by Culicoides, which may eventually lead to an outbreak of BTD. The state of West Bengal shares 6.9% of the total livestock population, of which 8.3% are cattle, 0.8% sheep, and 10.9% goats (20th Livestock Census. 2019). The BTV seroprevalence in goats (24.03%), sheep (30-40%), and cattle (16.21%) has been reported across 7 districts of West Bengal (Halder et al. 2016). Although anti-bluetongue (BT) antibodies have been detected in sheep, goats, and cattle, an outbreak was yet to be reported from West Bengal (Mandal et al. 2011). Since 1997, two thousand outbreaks of BTD have been recorded in India, predominantly in sheep. Although cattle, buffalo, and goats are prone to BTD, clinical manifestation was restricted (Prasad et al. 2008). However, the livestock act as a reservoir, infecting the Culicoides for subsequent transmission. Twenty-two serotypes of BTV have been reported from India, causing 0.4 million cases, resulting in 64,000 casualties (Rupner et al. 2020, Saminathan et al. 2020).

Worldwide, the association of Culicoides with the host was reported by Kettle (1969), Barnard and Jones (1980), Lillie et al. (1988), Zhang and Gerry (2023), Zhang et al. (2024) from the Nearctic; Hoch et al. (1990) from Neotropical and Blackwell (1997), Van der Rijt et al. (2008), Viennet et al. (2011, 2012), Sanders et al. (2012) from Palaearctic. Host-seeking activities of the Culicoides species mentioned in the present study were yet to be reported elsewhere. While forecasting disease outbreaks, limited information on the host–vector association has led to assumptions that all the Culicoides species prevalent in the cattle shed fed on the host with equal aggressiveness (Elbers and Meiswinkel 2015), leading to many misconceptions. Difficulty in aspiration of adult Culicoides from the host body also limits studies related to the host-seeking activities (Viennet et al. 2012).

Few studies deal with adult midges attacking farm animals outdoors (exophily) and indoors (endophily) (Viennet et al. 2011, Allyòn et al. 2014). In the Palaearctic region, for example, horses were housed to prevent the animals from the bite of Culicoides species spreading the African horse sickness virus (Meiswinkel et al. 2008, Baylis et al. 2010). In the eastern Indian state of West Bengal, most of the animal shelters are open type, and any information on these activities of the midges would suggest the risk of cattle being exposed to the vectors.

The present investigation assessed preferential landing on cattle, diel activity, biting rate, attack rate, and feeding success of livestock-associated vector species of adult Culicoides. The exophily and endophily of Culicoides were evaluated to substantiate the host-seeking activity. The species were identified morpho-taxonomically and validated with COX1-based molecular sequences submitted to NCBI.

Materials and Methods

Study 1: Host-seeking Activities of Culicoides

Study Sites

Trapping was done in 2 rural villages amidst agricultural fields where rice cultivation was practiced throughout the year. The sites were 51 km apart in Burdwan district, West Bengal (i) Dharan (DH; 23°04ʹ04.4″N, 87°83ʹ95.5″E) and (ii) Sahebganj-Tantipara (ST; 23°26ʹ01.0″N, 87°82ʹ79.8″E) (Fig. 1a, d). At DH, field studies were done in 2018 (from May to October), whereas at ST, field data were collected in 2018 (May to October) and 2019 (April to June). Collection was done in a single shed at DH (only aspirator was used) and 2 sheds at ST (one each for aspirator and light trap). The DH and ST sheds each housed a white cow from the Sahiwal crossbreed of Bos taurus (Fig. 1b, e, f). The sampling month and period followed Harsha et al. (2020), which stated that an abundance of Culicoides spp. associated with cattle sheds increased from April to October and gradually declined as winter set in. During the period of observation, sunset was between 17:13–18:12 h and sunrise between 04:52–05:35 h. The cattle sheds were made of mud bricks located in the household’s backyard. The study sites received 1,496 mm of annual rainfall (Rainfall Statistics of India 2018, 2019). The hygiene conditions were compromised within and outside the perimeter of the animal sheds due to the unorganized dumping of litter in dung heaps and paddy straws.

Description of Collection

The oral aspirator was used as a sampling device at both DH and ST sites and was conducted from 18:00 to 06:00 h. In the text, the following abbreviations have been used: sunset “ss” and sunrise “sr” and “– sign” represent before, and “+ sign” means after the sunset and sunrise. The catch period was segregated into 11, 1-h intervals: - T1 (-ss [sunset] 1hr) to T11 (+ sr [sunrise]), with 1 h excluded for farm management (i.e., 22:00–23:00 h). At ST, 12 collections were made, i.e., 2 collections per month. At DH, 15 collections were made, i.e., 3 collections per month, except for October (2018), April, and June (2019), where 1 collection per month was done. Twenty-seven collections were done at both sites, resulting in 297 hrs of collection.

Adult Culicoides landing on different body parts of cattle were collected by aspirator. For each collection, midges from the cattle were aspirated by a person. Thirty minutes before sampling, other cattle, except one, were moved out of the shelter. The midges were visually spotted and gently collected with a mouth aspirator from the different surface areas of the cattle, viz., head (H1), neck (H2), hump (H3), back (H4), leg (H5), belly (H6), and hip (H7) (Allyòn et al. 2014, Elbers and Meiswinkel 2015). Aspiration was performed for 10 min every hour. The aspirated adult Culicoides were immediately transferred to 70% EtOH. The adults were stored at −20°C in the laboratory for further sorting and DNA-based molecular work. Later, the catch data were further processed and analyzed to obtain the biting rate and attack rate of the midges. The temperature of different surface areas of cattle was measured using non-contact infrared digital temperature gun. The biting rate was calculated as the total number of engorged midges (E) collected per 10 min, and the attack rate was measured as total landings (L) per 10 min. A rechargeable white LED headlamp (3W, 2,000 mAh) was used during the collection to facilitate the easy visualization of midges on the cattle. At ST, 2 sheds were selected for collection which were 300 m apart. In one shed, the light trap was operated, whereas aspirator was used in another shed. In the laboratory, voucher specimens of species trapped during this investigation were kept aside for DNA extraction by a non-destructive method. After extraction, these voucher Culicoides specimens were mounted on glass slides for identification using the taxonomic key (Wirth and Hubert 1989) and molecular identification. After identification, the females were age-graded based on external abdominal pigmentation (Dyce 1969). Here, the unfed nulliparous and unfed parous were considered non-engorged, and the rest were engorged. The number of gravid females was also visually determined.

For comparative assessment, both the light trap and aspirator were operated simultaneously at site ST. UV LED LT (Mazumdar et al. 2019) was operated in a cattle shed at ST from 18:00 to 06:00 h and placed 6 feet from the cattle. The midges were caught every hour, which resulted in 11 collections per night (excluding one hour). A total of 12 nights of light trap-based sampling were done, with 2 nights per month resulting in 132 h of collections.

Study 2: Indoor–Outdoor Activity of Culicoides

Study Site

Field assessments were done at a peridomestic cattle farm located within the municipal limits of Memari town (23°10ʹ38″N; 88°06ʹ01″E), situated in the agricultural heartland of the district of Purba Burdwan, West Bengal, India (Fig. 1c). The animal shelters were located near a small water body bordered by vegetation, coconut trees and a dump yard of cow dung and leaf litter. The farm consisted of 3 sheds, housing 8 cattle; 2 sheds were the closed type with a single door and window, whereas the remaining one was an open type. During the daytime, the cattle were herded into open yards. For indoor assessment, 4 cattle were kept in one of the closed sheds, whereas the other closed shed remained vacant. Similarly, 4 cattle were placed in the open shed for outdoor assessment (Fig. 2).

Description of Collection

Four Ultra violet-Burdwan University-Light trap (UV BU LT) (Mazumdar et al. 2019) were used in this evaluation. These light traps were hung 2 m above the ground and switched on at dusk (between 17:30 and 18:00 h). Four light trap treatments were used: P1—outdoor, with cattle; P2—indoor, with cattle; P3—indoor, without cattle; and P4—outdoor, without cattle (Fig. 2). To minimize luminal interference, surrounding lights were switched off near the farm. The trapping duration was from dusk to dawn, resulting in a total of 264 collections over 33 nights between May to November 2016 [inclusive of pre-monsoon (May, June), monsoon (July, August), and post-monsoon (September to November)] months. Before the installation of the light trap, the cattle were moved in or out of their respective holds with care and the owner’s permission. The trapped insects were preserved in 70% EtOH and immediately transferred to the laboratory for sorting, age grading, and taxonomic identification of the specimens.

DNA Extraction and PCR Amplification

DNA was extracted from individual specimens of C. oxystoma, C. peregrinus, C. fulvus, and C. anophelis using a non-destructive method (Harrup 2018). PCR amplification (HiMedia Prima 96 plus) of the extracted DNA was done based on COX1 with the forward and reverse primers, LCO1490 (5ʹ-GGT CAA CAA ATC ATA AAG ATA TTG G-3ʹ) and HCO2198 (5ʹ-TAA ACT TCA GGG TGA CCA AAA AAT CA-3ʹ) (Folmer et al. 1994). The primers were purchased from Eurofins Pvt. Ltd., Bengaluru, India. The adult Culicoides were placed in a lysis buffer for DNA extraction. The composition of the lysis buffer (for 10 ml) was 1 ml Tris HCL (1M), 0.4 ml NaCl (5M), 0.2 ml SDS 10% (w/v) in 8.2 ml nuclease-free water (HiMedia TCL086), 100 µl EDTA, and 100 µl Proteinase K (HiMedia MB086). PCR was done with 25 µl of Taqmixture (Himedia, MBT061) having 0.5 pmol of forward and reverse primer, 6 µl of nuclease-free water (Himedia TCL016), and 5 µl of sample DNA. Samples were Sanger sequenced using the same primer (GeneSpec Pvt. Ltd., Kochi, India). Raw sequences were trimmed and consensus sequences were obtained from forward and reverse sequences by using BioEdit (ver. 7.7.1). Sequences were aligned using CLUSTALW in MEGA-X.2.6. The results were matched and submitted to NCBI. Because the DNA was obtained through a non-destructive process, the voucher specimens were dissected and slide mounted following Wirth and Marston (1968) for morphological identification.

Statistical Analysis

Host-seeking Activity and Comparison of Trapping Methods

A logistic regression was applied to the data on the successful and unsuccessful attempts to feed made by the female Culicoides to assess the effects of time and site on host landing by the species-specific age group. A binomial generalized linear model with a logit link was used, considering time and site as the explanatory variables. The logistic regression equation was of the form: (y) = 1/(1 + exp(-(a + b₁ × ₁ + b₂ × ₂ + b₃ × ₃)), where y was the response variable, and the explanatory variables, x₁, x_2, and x_3, represented the landing time on host, landing site on host, and the interaction between the 2 components, respectively. The regression analysis was performed with the assumptions that the landing of non-engorged and engorged Culicoides follow a binomial distribution (n, p) with n replicates for each set of independent variables (time of landing, site of landing on host and interaction between host landing and time). After aligning the data in binomial order, the analysis was done to interpret the relation between successful and unsuccessful feeding age groups preferring particular landing sites on cattle bodies. A maximum likelihood method was used to estimate the logit-linked parameters through the statistical software XLSTAT (Addinsoft 2010). Using the value of Wald’s Chi-square, the parameters of the models were tested for significance at P = 0.05 level (McCullogh and Nedler 1983). The ratio between the numbers of engorged Culicoides (E) and the total number of Culicoides landed (L) was used to calculate the feeding success at each time point. The success of feeding at time T_n = No. of engorged (E) at T_n/Total no. midges landed (L) on the host body at T_n-1. The data were represented as specimens per minute attacking or biting the host. All the graphs were presented using the Origin Pro 8.0 software.

Indoor–Outdoor Activity

The numbers of trapped adults were subjected to mixed model ANOVA with the month (seasonal), time (photoperiod), and outdoor–indoor location of the host (location) as explanatory variables using XLSTAT software (Addinsoft 2010). The variability of the midge population was represented as a function of the photoperiod, which accounted for diurnal variations, and the month, which accounted for the seasonal variability. The catch data consists of 264 collections of the variability of the midges at spatial and temporal scales, inclusive of the short-term and long-term time variability. The effect of the host’s location (explanatory variable) was assessed as mixed model ANOVA followed by a post hoc Tukey test considering the relative abundance of Culicoides as a response variable. Based on dissimilarities in the abundance of different Culicoides midges and the landing location (marker of exophily and endophily), a non-metric multidimensional scaling (NMDS) was carried out. A proximity matrix derived from the dissimilarity based on the Euclidean distances followed by Ward’s method (Legendre and Legendre 1998) was used as an input for the construction of the biplot for the NMDS. The proportions of parous individuals sampled among the 4 experimental setups (outdoor, with cattle [P1]; indoor, with cattle [P2]; indoor, without cattle [P3]; outdoor, without cattle [P4]) were subjected to ANOVA with the post hoc Tukey test.

Results

Host-seeking Activity

A total of 8,758 Culicoides were collected using aspirator and 6,698 individuals were collected in light trap across both sites. At DH, a total of 3,994 female individuals were collected by aspirator. Female Culicoides oxystoma comprised 48% of the catch, of which 32% were engorged. Female Culicoides peregrinus comprised 52%, of which 42% were engorged. At ST, a total of 4,764 females were collected by aspirator. Culicoides oxystoma comprised 44% of individuals, of which 42% were engorged. Culicoides peregrinus constituted 56% of individuals, of which 62% were engorged.

A significant number of Culicoides individuals landed on the upper portion of the cattle (H1, H2, and H3), while few landed on the rest of the body (H4, H5, H6, and H7). The actual feeding time of C. oxystoma and C. peregrinus on the cattle was between 4:00 h and 5:00 h (T10, -sr 1 h) with a duration of 30–40 min (Fig. 3). The females swarmed over the host before attacking the cattle. The feeding success ranged between 29.66% and 42.40% for C. oxystoma and 45.43% to 50.19% for C. peregrinus at both sites. At DH, the feeding success of both species was high (above 50%) at T8 (-sr 3 hr), T9 (-sr 2 hr), and T10 (-sr 1 hr). At ST, both the species exhibited successful feeding at T6 (-sr 5 hr), T9 (-sr 2 hr), and T10 (-sr 1 hr). Culicoides oxystoma also exhibited successful feeding at T3 (+ ss 3 hr), whereas C. peregrinus also exhibited successful feeding at T2 (+ ss 2 hr).

GLM results showed significant differences in the landing time and site of Culicoides landing on the host by non-engorged and engorged females. The females that successfully fed were designated as engorged and those who attempted to feed were mentioned as non-engorged (Table 1). T-test results implied equal feeding propensity (t = −1.05, P > 0.05) between C. oxystoma and C. peregrinus. The biting rate of C. oxystoma ranged between 0.02–3.96 midges per minute, and C. peregrinus was 0.01–7.99 midges per minute. The attack rate of C. oxystoma was 0.02–11.11 midges per minute and for C. peregrinus 0.03–14.19 midges per minute. At both the sites, biting and attack rates reached the maximum at T10 (-sr 1 h).

Comparison of Light Trap vs Aspirator Trapping Methods

At the ST site, light traps represented 57.60% of Culicoides collected, compared to 42.39% by aspirator (Fig. 4; Supplementary Table 1). Culicoides oxystoma was prevalent in light trap catch during T1, T2, T4, and T8 to T11, whereas aspirator catch data showed peak activity at T10 (Fig. 3). Likewise, light trap collections of C. peregrinus were statistically significant at T1 to T4 and T8 to T11 compared to other time periods, whereas, the aspirator catch exhibited maximum landing and biting activity at T10 followed by T9 and T11. The light trap and aspirator collections showed that C. peregrinus was absent from T5 to T7, whereas the numbers of C. oxystoma were negligible during the same period.

Indoor–Outdoor Activity

A total of 21,105 adults were trapped indoors and outdoors, of which 1766 were males (8.4%), and 19,339 were females (91.6%). 31.23% of females were unfed nulliparous, 6.25% unfed parous, 60.14% engorged, and 1.90% gravid (Table 2). Five species were collected: C. oxystoma, C. peregrinus, C. fulvus, C. innoxius, and C. anophelis. The most abundant species was C. oxystoma (44.31%), followed by C. peregrinus (28.86%), C. fulvus (21.6%), C. anophelis (2.8%), and C. innoxius (2.4%). Light trap catches were significantly higher in the open shed in the presence of cattle (P1). Catch percentages in P1 were maximum (77.2%), followed by P4 (15.5%), P2 (5.6%), and P3 (0.8%). The male-to-female ratio was high in P1 (cattle placed outdoors) compared to P2 (cattle placed indoors), P3 (indoor location without cattle), and P4 (outdoor location without cattle). In P4, the male-to-female ratio was almost equal. A significant difference was observed in collection data between P1 and P2, vs P3 and P4 when subjected to mixed model ANOVA followed by post hoc Tukey test for C. oxystoma (F₃ = 47.53, P < 0.001), C. peregrinus (F₃ = 68.83, P < 0.001), C. innoxius (F₃ = 13.35, P < 0.001), C. fulvus (F₃ = 19.46, P < 0.001), and C. anophelis (F₃ = 16.52, P < 0.001). The proportion of male to female Culicoides and species-wise variation at different experimental treatments (P1, P2, P3, and P4) was analyzed using ANOVA with post hoc Tukey test: F₃ = 60.32, P < 0.0001. The male and female proportions were almost equal, near probable breeding habitat (P4); however, the female proportion increased in the cattle sheds. We observed that the parity of the midges differed significantly among the 4 treatments (F₃ = 4.12, P < 0.005). Significant differences among different species were also noted (F₃ = 17.71, P < 0.0001).

The numbers of parous individuals differed significantly between P1 (cattle located outdoors), P3 (indoor collections in the absence of cattle), and P4. Based on NMDS, 4 situations in the experimental design have been clustered into 2 subsets; subset 1 consisted of P1 (cattle positioned outdoors) and P4 (collections in probable breeding habitat in the absence of cattle); subset 2 has P2 (indoor sampling of midges in the presence of cattle) and P3 (indoor sampling of midges in the absence of cattle). The biplot depicts the ordination of the host location in the study area where Kruskal’s stress value remained far from unity (0.039), justifying the use of the statistics (Fig. 5).

Molecular Identification of the Species

The sequences of the species obtained were submitted in NCBI under the provided accession numbers. C. oxystoma (PP833148 and PP833149) showed 99.53% similarity with OM049245 (collection site: Yelahanka, Karnataka, India), and 99.43% similarity with KT307839 (collection site: Kancheepuram, Tamil Nadu, India), respectively. C. fulvus (PP833147) showed a similarity of 99.05% with KT352643 (collection site: Kautru, Papua New Guinea). C. peregrinus (PP833150 and PP833151) showed 100% similarity with KT307848 (collection site: Kalapatti, Tamil Nadu, India). C. anophelis (PP833152 and PP833153) showed 100% similarity with KT307787 (collection site: Madhavaram, Tamil Nadu, India).

Discussion

Host-seeking Activity and Comparison of Trapping Methods

Studies on association of Culicoides with its host were done on the following species: Culicoides furens, Culicoides barbosai, Culicoides mississippiensis, Culicoides paraensis, Culicoides impunctatus, Culicoides brunnicans, Culicoides obsoletus, Culicoides scoticus, and Culicoides dewulfi (Kettle 1969, Barnard and Jones 1980, Lillie et al. 1988, Hoch et al. 1980, Blackwell 1997, Sanders et al. 2012, Viennet et al. 2012). All the studies remarked that diel activity and host-seeking pattern are relative to the host location, host composition, and seasonal influences. The Culicoides species investigated in this study were reported as probable vectors of BTV in India (Harrup et al. 2016). Shielding animals from attacks by female Culicoides may be adopted to break the chain of disease transmission; hence, information on the peak activity of Culicoides from this region will be helpful in management strategies (Harsha et al. 2020). The landing time of C. oxystoma and C. peregrinus females was early morning to obtain a blood meal, and their preferred site was the hump and neck regions of cattle. Aspirator-based interception of blood-seeking C. oxystoma and C. peregrinus to hosts depicts diel activity similar to observations implications made by Lillie et al. (1988). Viennet et al. (2011) further substantiated this, as gravid females and males were absent in the aspirator collections. Cattle as hosts attracted C. oxystoma and C. peregrinus. Both vector species were prevalent in the cattle sheds of West Bengal, as was previously reported by Harsha et al. (2020). A significant proportion of engorged C. peregrinus and C. oxystoma in the cattle suggests active blood-seeking behavior, which supports the fierceness of C. peregrinus and C. oxystoma as recorded by Sukarsih et al. (1996). The species’ biting and attack rates appeared alike, implying a high feeding success over a short span. However, the subtle differences in attack and bite rate depict that a mere landing on the host does not warrant feeding. Although light trap catch data validated the crepuscular prevalence of the midges, the most preferred feeding window of C. oxystoma and C. peregrinus on cattle was an hour before sunrise. Dusk and dawn are thought to be the preferred feeding times of Culicoides, with behavior triggered by sudden changes in light intensity (Hoch et al. 1990, Viennet et al. 2012). Nonetheless, Culicoides are prevalent in resting conditions in the cracks, crevices, walls, and roofs within the shed housing cattle throughout the 24-h period (Mondal et al. 2022). The proportion of C. oxystoma and C. peregrinus individuals feeding successfully on cattle depends on the abundance of midges attacking the cattle, as opined by Van der Edyne et al. (2021). We recorded 45% of the total number of midges landing had fed on the cattle, similar to other studies (e.g., 40.3% in Townley et al. [1984]). The absence of C. fulvus in aspirator may be attributed to the species’ population being sufficiently low near the study area, or having a lower preference for cattle than C. oxystoma and C. peregrinus.

The landing sites of C. oxystoma and C. peregrinus were the neck and hump followed by the head of the cattle (mostly in and around the ear). Although various studies suggested temperature to be a significant factor influencing the landing of Culicoides (Braverman 2008), skin thickness and vascularization may be other determining factors. There was a difference of 1°C in temperature between the belly and hip of cattle which was measured during the study. However, in this study, there were fewer Culicoides on the belly compared to the neck and hump, which may be due to varying degrees of skin thickness regionally differentiated at different body parts of the host (Lehane et al. 2005). The thickness of the skin in the neck region of cattle is relatively less (~3.3 mm) compared to an average of 4.2 mm of the rest of the body (Hamid et al. 2000). Adult female Culicoides possess short and stout mouthparts; preferably feeding on skin areas rich in vascularization (Lehane et al. 2005). However, variations in preferential landing are a species-specific attribute and vary with the host. Culicoides brunnicans and C. dewulfi preferred the upper part of the head and back of sheep, whereas C. obsoletus preferred the lower part (Viennet et al. 2011). Elbers and Meiswinkel (2015) reported that C. chiopterus favored legs, while C. punctatus, and C. acharya landed on the belly in a comparative preferential landing involving cows, sheep, and a Shetland pony. In the same study, C. obsoletus, C. dewulfi, and C. pulicaris landed on the head, back, and flanks, respectively. Moreover, C. chiopterus, C. punctatus, and C. obsoletus/scoticus favored the belly region of horses (Elbers et al. 2016).

Indoor–Outdoor Activity

The difference in outdoor and indoor prevalence of midges in the presence and absence of cattle was ascertained. Cattle in open sheds were prone to midges’ attack as most engorged Culicoides were collected outdoors. The blood engorged proportion of C. peregrinus was high. Species abundance, composition, and age grading were similar at dusk and dawn. The exophilic nature of these species increases the probability of BTV transmission when cattle are placed outdoors, as stated by Meiswinkel et al. (2008). Cattle sheltered inside a closed shed were less prone to midge bite. Similar observations were made by Baylis et al. (2010) on Culicoides of Northern Europe. Meiswinkel et al. (2008) remarked on the exophilic nature of the Culicoides as 3-fold more were collected outdoors than indoors. In this study, an abundance of Culicoides increased by 6 times in the presence of cattle, irrespective of their position, and outdoor collection was 14 times more than indoor collection. However, the indoor–outdoor activities of Culicoides vary from species to species.

The presence of nulliparous individuals in light trap catches signifies the proximity of probable breeding habitat which was also the location for the operation of trap outdoor in absence of cattle. A high female-to-male ratio was observed in the traps operated close to cattle, which further decreased as the light trap was shifted near the probable habitat (away from the cattle). The occurrence of < 5% gravid females in the catch data suggests that the population might be exiting the farm after blood feeding for oogenesis in a different location (Meiswinkel et al. 2008), or the age group is less attracted to the light trap. However, a possible conclusion requires further introspection.

Conclusion

In India, cattle are housed in either open sheds or in open yards, leading to continuous exposure of cattle to bites of Culicoides. Animal husbandry in such conditions makes the present work relevant. Two studies were undertaken in different regions of rural West Bengal to exhaustively assess the host-seeking activities of the Culicoides species associated with cattle. The aspirator-based collection showed that C. peregrinus and C. oxystoma inflict bites on cattle early in the morning (before sunrise). We opine that the farmers should house the cattle in a “closed enclosure” and cover the “neck” and “hump” region of cattle. Furthermore, a comparison between light trap and aspirator-based collections was also made. The light trap collection exhibited the crepuscular (dusk and dawn) prevalence of Culicoides in cattle sheds. Culicoides oxystoma, C. peregrinus, C. fulvus, and C. anophelis, exhibited exophily on cattle farms.

Supplementary material

Supplementary material is available at Journal of Medical Entomology online.

Acknowledgments

The authors acknowledge the Head of the Department of Zoology at the University of Burdwan for providing the study facility. Grammarly Premium AI tool has been used for proofreading. The BioRender tool has been used to create illustrations. We are indebted to Dr. Glenn Bellis, Charles Darwin University, Australia, for helping in species identification by providing relevant literature.

Author contributions

Shuddhasattwa Maitra Mazumdar (Conceptualization [Lead], Data curation [Lead], Formal analysis [Lead], Investigation [Lead], Methodology [Lead], Writing—original draft [Lead]), Nabanita Banerjee (Investigation [Equal], Methodology [Equal]), Biswajit Mondal (Investigation [Equal], Methodology [Equal]), Arjun Pal (Investigation [Equal]), Surajit Kar (Methodology [Equal]), Rupa Harsha (Writing—review & editing [Equal]), and Abhijit Mazumdar (Project administration [Equal], Supervision [Equal], Validation [Equal], Writing—review & editing [Equal])

Funding

No financial grant was received for the study.

Ethical Statement

No animals were harmed during the study.

Consent to Participate

All authors made contributions toward the execution of the present study. All the authors approved the manuscript.

Data Availability

Information supporting the findings of the study is available in the manuscript.

References