The biting midges, Culicoides peregrinus Kieffer and Culicoides oxystoma Kieffer (Diptera: Ceratopogonidae) are the most significant vector species of bluetongue virus (BTV) in the Oriental region, including India. Rearing of these vector species was cumbersome; previous researchers supplemented the rearing substrates primarily with cattle dung (the habitat), yeast, and nutrient broth. Other investigations reiterated that an enriched milieu of live bacteria is required for the oviposition and developmental progression of the immatures, as they failed to develop in sterile medium. Therefore, bacteria-based approaches provide novel opportunities for artificial rearing. This investigation tries to simplify and create a cleaner version of rearing based on different bacterial strains. The substrate bacterial strains were biochemically characterized, and their influence on oviposition, hatching, and larval development was analyzed and evaluated under laboratory conditions. We artificially reared two vector species by utilizing three different strains of Bacillus cereus and one strain of Alcaligenes faecalis retrieved from the substrates. The results demonstrated that gravid females select their oviposition substrates based on stimuli derived from live microorganisms that indicate the suitability of the developmental substrate for immature development. Bacillus cereus 1B stimulated the greatest extent of egg hatching (>99%), larval survivability (>74%), Pupae formation (>83%), and adult emergence (>98%) in both species. This present investigation proposes to utilize the B. cereus 1B as an alternative approach to artificially rear and establish laboratory colonies of these vector species.

Live adult midges were collected into a muslin cage fitted under the UV LED light trap operated overnight in a backyard cattle shed located in the district of Burdwan (West Bengal, India). The live engorged females of Culicoides spp. were captured in autoclaved small glass vials. The engorged females of C. peregrinus and C. oxystoma were identified by observing the glass vials containing midges under a binocular microscope.

The oviposition bed was prepared by placing autoclaved absorbent cotton at the bottom of an autoclaved glass vial, and a round piece of filter paper was placed on top of it. The beds were moistened with respective cultures of the following pure-cultured bacterial strains (~ 10² CFU per mL): Bacillus cereus 1B, B. cereus MFA, B. cereus MFB, and A. faecalis CU5A, which were allowed to grow for 48 hours in nutrient broth medium (HiMedia, M002) solution. The following experimental controls were considered: oviposition beds moistened with a) distilled water and tap water (both autoclaved) as the negative control, and b) distilled water and tap water (non-autoclaved) as the positive controls for C. peregrinus and C. oxystoma, respectively. The vials were examined daily for oviposition. Simultaneously, the abdomens of the females were dissected to look for any leftover eggs. The oviposited eggs of C. peregrinus and C. oxystoma were counted under a binocular microscope and transferred to the rearing plates for further hatching and larval rearing.

The rearing plates were prepared by using autoclaved Petri dishes containing autoclaved absorbent cotton moistened with bacterial cultures consisting of Bacillus cereus 1B, B. cereus MFA, B. cereus MFB, and A. faecalis CU5A, respectively. These cultures were allowed to grow for the next 48 hours in the nutrient broth medium. The following experimental controls were considered: rearing plates prepared with a) autoclaved substrate (mud-broth and yeast solutions) as a negative control, and b) non-autoclaved (mud-broth and yeast solutions) as positive controls for C. peregrinus and C. oxystoma, respectively.

The influence of bacterial strains on oviposition and retention of eggs within their abdomens was analyzed by one-way ANOVA followed by the Tukey HSD test (p<0.05). The different life history parameters, egg hatching, survivability of larvae, pupae, and adult emergence of the reared C. peregrinus and C. oxystoma, in different rearing substrates containing bacterial strains, were analyzed using PERMANOVA. Significant differences in the number of hatched eggs, larvae, pupae, and emerged adults across different bacterial strains were noted, followed by a pairwise test (p<0.05).