DOI: 10.5061/dryad.8931zcs3z
title: Influence of bacterial strains on oviposition and larval development of two BTV vector species of Culicoides (Diptera: Ceratopogonidae): an approach to colony establishment

description: 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
 (&gt;99%), larval survivability (&gt;74%), Pupae formation
 (&gt;83%), and adult emergence (&gt;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.
description: 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 <em>Culicoides</em> spp. were captured in autoclaved small
 glass vials. The engorged females of <em>C. peregrinus</em>
 and <em>C. oxystoma</em> 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 (~
 10<sup>2</sup> CFU per mL): <em>Bacillus
 cereus</em> 1B, <em>B. cereus</em> MFA, <em>B.
 cereus</em> MFB, and <em>A. faecalis</em> 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 <em>C.
 peregrinus</em> and <em>C. oxystoma</em>, 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 <em>C. peregrinus</em> and <em>C.
 oxystoma</em> 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 <em>Bacillus cereus</em>
 1B, <em>B. cereus</em> MFA, <em>B. cereus</em>
 MFB, and <em>A. faecalis</em> 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 <em>C.
 peregrinus</em> and <em>C. oxystoma</em>,
 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
 (<em>p</em>&lt;0.05). The different life history
 parameters, egg hatching, survivability of larvae, pupae, and adult
 emergence of the reared <em>C. peregrinus</em> and
 <em>C. oxystoma</em>, 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 (<em>p</em>&lt;0.05).
description: # Influence of bacterial strains on oviposition and larval development of
 two BTV vector species of Culicoides (Diptera: Ceratopogonidae): an
 approach to colony establishment Dataset DOI:
 [10.5061/dryad.8931zcs3z](10.5061/dryad.8931zcs3z) ## Description of the
 data and file structure The raw data are in .xlsx format, containing data
 of abdominal eggs, number of oviposited eggs, hatched eggs, larval, pupal
 survivability, and adult emergence of *Culicoides peregrinus* and
 *Culicoides oxystoma* when reared by utilizing 4 bacterial strains. The
 following experimental controls were considered for oviposition:
 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 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. One data file contains
 information on abdominal eggs (retained eggs within the abdomens) of
 *Culicoides peregrinus* and *Culicoides oxystoma* when reared by utilizing
 4 bacterial strains (*B. cereus* 1B, *B. cereus* MFA, *B. cereus* MFB, *A.
 faecalis* CU5A) as well as on oviposition beds taken as negative and
 positive controls. The other file contains the number of oviposited eggs,
 hatched eggs, larval, pupal survivability, and adult emergence of
 *Culicoides peregrinus* and *Culicoides oxystoma* when reared by utilizing
 4 bacterial strains (*B. cereus* 1B, *B. cereus* MFA, *B. cereus* MFB, *A.
 faecalis* CU5A) as well as on oviposition beds and rearing substrates
 taken as negative and positive controls. #### File:
 Data_on_abdominal_eggs.xlsx **Description:** The raw data are in .xlsx
 format, containing data of abdominal eggs of *Culicoides peregrinus* and
 *Culicoides oxystoma* when reared by utilizing 4 bacterial strains. The
 following experimental controls were considered for oviposition:
 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. #### File:
 Data_on_oviposited_eggs_and_rearing.xlsm **Description:** The raw data are
 in .xlsx format, containing data on the number of oviposited eggs, hatched
 eggs, larval, pupal survivability, and adult emergence of *Culicoides
 peregrinus* and *Culicoides oxystoma* when reared by utilizing 4 bacterial
 strains. The following experimental controls were considered for
 oviposition: 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 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. ## Code/software Past
 v4.07 and RStudio v4.2.2 software were used.