Laboratory Rearing of Culicoides stellifer (Diptera: Ceratopogonidae), a Suspected Vector of Orbiviruses in the United States

Abstract

Laboratory rearing procedures of Culicoides stellifer Coquillett (Diptera: Ceratopogonidae) were evaluated with an aim towards colonization of this species. Eggs collected from field-collected gravid females were placed on 0.25% agar slants and given a diet of 1) nematodes (Panagrellus redivivus Linnaeus), 2) nematodes + lactalbumin and yeast (LY), 3) microbes from nematode medium, and 4) tap water (autoclaved). Complete larval development to adult stage occurred only in two treatments: 1) nematodes and 2) nematodes + LY. Culicoides stellifer larvae could not survive beyond 1 wk on a diet of microbes alone or in the sterile water treatment. Larval survival rates were high using nematode diet (79.2 ± 11.3% [mean ± SE]) but were slightly lower in the nematode + LY group (66.5 ± 19.6%). Larval stage lasted ~21 d in both treatments. Sex ratio of F1 adults was ~1:1 (M:F) using nematode diet but was male biased (~2:1) with nematode + LY diet. These findings collectively suggest that a microbial community is required for midge larvae, either to support invertebrate prey base or as a potential food source. But in the present study, the supplied microbes alone were not sufficient to support midge survival/development. It appears that other nutritional components may also be essential to support the larval survival/development of C. stellifer. Overall, a simple diet of bacterial feeding nematodes and their associated microorganisms can be used to rear C. stellifer larvae under laboratory conditions. However, captive mating in F1 adults poses a major obstacle for successful colonization of this species currently.

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Development, Life History
Laboratory Rearing of Culicoides stellifer (Diptera:
Ceratopogonidae), a Suspected Vector of Orbiviruses in
the UnitedStates
Dinesh Erram1 and Nathan Burkett-Cadena
Florida Medical Entomology Laboratory, University of Florida, IFAS, 200 9th St. SE, Vero Beach, FL 32962, Phone: (772)778-7200, Fax:
(772)778-7205 (derram@ufl.edu; nburkettcadena@ufl.edu) and1Corresponding author, e-mail: derram@ufl.edu
Subject Editor: Lane Foil
Received 3 June 2019; Editorial decision 14 August 2019
Abstract
Laboratory rearing procedures of Culicoides stellifer Coquillett (Diptera: Ceratopogonidae)were evaluated with
an aim towards colonization of this species. Eggs collected from field-collected gravid females were placed on
0.25% agar slants and given a diet of 1)nematodes (Panagrellus redivivus Linnaeus), 2) nematodes + lactal-
bumin and yeast (LY), 3) microbes from nematode medium, and 4)tap water (autoclaved). Complete larval
development to adult stage occurred only in two treatments: 1)nematodes and 2)nematodes + LY. Culicoides
stellifer larvae could not survive beyond 1wk on a diet of microbes alone or in the sterile water treatment.
Larval survival rates were high using nematode diet (79.2± 11.3% [mean ± SE]) but were slightly lower in the
nematode + LY group (66.5± 19.6%). Larval stage lasted ~21 d in both treatments. Sex ratio of F1 adults was
~1:1 (M:F) using nematode diet but was male biased (~2:1) with nematode + LY diet. These findings collectively
suggest that a microbial community is required for midge larvae, either to support invertebrate prey base or
as a potential food source. But in the present study, the supplied microbes alone were not sufficient to support
midge survival/development. It appears that other nutritional components may also be essential to support the
larval survival/development of C.stellifer. Overall, a simple diet of bacterial feeding nematodes and their as-
sociated microorganisms can be used to rear C.stellifer larvae under laboratory conditions. However, captive
mating in F1 adults poses a major obstacle for successful colonization of this species currently.
Key Words: Culicoides stellifer, biting midges, Orbiviruses, laboratory rearing, colonization
Culicoides species (Diptera: Ceratopogonidae), commonly referred
to as biting midges or no-see-ums, transmit numerous disease-
causing agents to vertebrates including humans worldwide (Mellor
etal. 2000, Pfannenstiel et al. 2015). Among the several pathogen
classes Culicoides species transmit, bluetongue virus (BTV) and epi-
zootic hemorrhagic disease virus (EHDV) (Genus Orbivirus, Family
Reoviridae) are of major concern because these viruses affect a va-
riety of domestic and wild ruminants, thus exerting a signicant ec-
onomic impact on animal agriculture worldwide (Mellor etal. 2000,
Pfannenstiel etal. 2015). In North America, Culicoides sonorensis
Wirth and Jones and Culicoides insignis Lutz are currently the only
conrmed vectors of BTV and/or EHDV (Foster etal. 1977, Jones
et al. 1977, Tanya etal. 1992, Tabachnick 1996). However, other
vector species are likely to exist, particularly in the southeastern
United States, where the two conrmed vectors of Orbiviruses are rare
(Mullen etal. 1985; Smith and Stallknecht 1996; Smith etal. 1996;
Ruder etal. 2015; McGregor etal. 2019a, 2019b). Some Culicoides
species suspected to be involved in BTV/EHDV transmission in
this region are Culicoides stellifer Coquillett, Culicoides venustus
Hoffman, and Culicoides debilipalpis Lutz among many others.
Unfortunately, no effective midge control strategies exist currently,
primarily because many of the fundamental aspects of Culicoides
species associated with Orbivirus transmission, including their bi-
ology and ecology are poorly understood. As such, colonization of
Culicoides species pertinent to BTV/EHDV transmission provides
a valuable resource to accelerate research towards understanding
various fundamental aspects of these vectors including vectorial ca-
pacity, vector competence, vector-virus-host interactions, and the
ecology of Orbiviruses. Currently, C.sonorensis represents the only
colonized conrmed vector of Orbiviruses worldwide (Jones etal.
1969). Therefore, colonization of C. stellifer and other Culicoides
species involved in BTV/EHDV transmission will be highly advan-
tageous in facilitating further research on various basic and applied
aspects of Orbivirus transmission in North America.
The agar and nematode method has been shown to be a useful
and convenient method for the larval rearing of Culicoides species
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Journal of Medical Entomology, XX(X), 2019, 1–8
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Research
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(Kettle et al. 1975). Using this method, several Culicoides species
have been reared in the laboratory previously including C.sonorensis
(Mullens and Velten 1994), Culicoides imicola Kieffer (Veronesi
et al. 2009, Barceló and Miranda 2018), Culicoides obsoletus
Meigen (Boorman 1985, Barceló and Miranda 2018), Culicoides
furens Poey (Koch and Axtell 1978), Culicoides melleus Coquillett
(Koch and Axtell 1978), Culicoides hollensis Melander and Brues
(Koch and Axtell 1978), and others (Kettle et al. 1975, Kitaoka
1982, Mullens etal. 1997, Barceló and Miranda 2018). However,
in many of these studies, midge larval rearing attempts for poten-
tial colonization purposes were evidently unsuccessful, primarily be-
cause sex-ratios of the F1 adults were found to be heavily distorted.
For example, the F1 adult sex-ratios were male-biased in C.imicola
(Veronesi et al. 2009, Barceló and Miranda 2018), C. obsoletus
(Boorman 1985, Barceló and Miranda 2018), Culicoides aterinervis
Tokunaga (Kitaoka 1982), and Culicoides newsteadi Austen (Barceló
and Miranda 2018), whereas the F1 sex-ratio was female-biased
in Culicoides matsuzawai Tokunaga (Kitaoka 1982), Culicoides
cataneii Clastrier (Barceló and Miranda 2018), and Culicoides
paolae Boorman (Barceló and Miranda 2018), with nondistorted F1
sex-ratios found only in Culicoides circumscriptus Kieffer (Barceló
and Miranda 2018). In this study, we evaluated laboratory rearing
procedures of C.stellifer, a strong suspected vector of BTV/EHDV
in North America. Culicoides stellifer is distributed throughout most
of United States between 49°N and 25°N latitudes and is one of
the most common species in the eastern United States (Blanton and
Wirth 1979). This midge species blood feeds heavily on white-tailed
deer (a highly susceptible vertebrate host of BTV/EHDV), has been
found to be naturally infected with EHDV, and has historically been
strongly associated with Orbivirus outbreaks in white-tailed deer,
particularly in the southeastern United States (Smith and Stallknecht
1996; Smith etal. 1996; Ruder etal. 2015; McGregor etal. 2019a,
2019b). Therefore, colonization of C. stellifer is advantageous,
which would tremendously help research efforts on understanding
various fundamental aspects of Orbivirus transmission, including
the potential establishment of effective vector management strat-
egies in North America. Thus, with a main aim towards colonization
of this species, we examined the larval development of C.stellifer
on agar slants using a diet of 1)nematodes, 2)nematodes supple-
mented with lactalbumin and yeast, 3)microbes from the nematode
medium, and 4)autoclaved tapwater.
Materials and Methods
Egg Collection From Field-CollectedMidges
Eggs of C.stellifer were collected from live midges that were trapped
using CDC-UV-LED traps setup at a commercial cervid farm in
Quincy, Gadsden County, FL (Erram and Burkett-Cadena 2018).
The eld-collected C. stellifer midges were separated from the
by-catch, blood fed on a live chicken, and were allowed to oviposit
on substrates using procedures described by Erram and Burkett-
Cadena (2018).
Larval Rearing Substrates
Larval rearing substrates used in this study were adapted from
Koch and Axtell (1978) and Kettle etal. (1975). Agar slants (0.25%
[w/v]) were prepared by boiling/dissolving 0.25g of agar (catalog #
BP1423-500, Fisher BioReagents, Atlanta, GA) in 100-ml tap water,
cooled to ~50°C temperature, poured onto one half of plastic Petri
dishes (60× 15mm, Fisherbrand, Atlanta, GA), and allowed to so-
lidify. The Petri dishes were inclined so that the agar poured would
result in a gentle slope (~15°). After solidication of agar, the other
half of the Petri dish was lled with tap water to a premarked level
such that a major portion of the agar surface was above the water
level (Fig. 1). The constant presence of tap water in the dish prevents
agar from drying and also serves as a medium where nematodes
or other diets are added/dispensed (Fig. 1). The setup of the larval
rearing substrate simulates the edge of a puddle habitat (but with the
mud replaced with agar) where C. stellifer adults were previously
found to mainly emerge from on the commercial cervid farm (Erram
and Burkett-Cadena 2018, Erram etal. 2019).
Larval Rearing Experiments
Eggs of C. stellifer (24–36 h old) were placed on the agar slants
and allowed to hatch (Fig. 1). Larval development of C.stellifer was
examined using four diets: 1)nematodes (~2.0mg; Panagrellus red-
ivivus Linnaeus(Rhabditida: Panagrolaimidae), Carolina Biological
Supply Company, Burlington, NC), 2) nematodes + Lactalbumin
and Yeast (LY, 1:1 ratio; ~2.0mg nematodes + ~2.0mg LY), 3)mi-
crobes from the nematode medium (104 CFU/ml; nematodes sep-
arated from the medium by suspending in tap water and ltering
using a Whatman lter paper funnel), and 4)tap water (autoclaved).
Diets under each treatment group were replenished with approxi-
mately the same amount every Monday, Wednesday, and Friday. In
addition, standing water levels in the dishes were maintained con-
stant throughout the study by adding tap water whenever necessary.
Developmental responses of C.stellifer recorded were egg stage du-
ration, egg hatch rate, larval survival to pupal stage, larval stage du-
ration, pupal stage duration, adult eclosion rate, and sex-ratio of
the emerging adults. Each dish had 10–25 eggs with three to ve
replicates per treatment and three independent trials were conducted
with each diet (Table 1). Laboratory conditions where the larval
dishes were incubated were 26± 1°C, 60–80% RH, and 14:10 (L:D)
h photoperiodcycle.
Statistical Analysis
Variation in egg hatch rates, larval survival rate to pupal stage, larval
stage duration, pupal stage duration, eclosion rates, and sex ratios
between trials within the same treatment group were analyzed using
generalized linear models (GLM) under binomial or negative bino-
mial distributions. Larval developmental responses recorded were
not compared between different treatment groups because these
experiments were not conducted at the same time. All data were
analyzed using R statistical software v.3.6.1 (R Core Team 2019)
using the packages MASS (Venables and Ripley 2002), car (Fox and
Weisberg 2011), and lsmeans (Lenth 2016).
Results and Discussion
Overall, the agar and nematode method proved to be a simple and
useful method for the larval rearing of C. stellifer. Agar serves as a
Fig. 1. Rearing of C.stellifer larvae on agar slants.
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good substitute for mud and also as a suitable larval rearing substrate
where C.stellifer larvae can be seen burrowing/moving through freely,
occasionally swimming into the standing water, and moving back into
the agar. The agar also serves as a convenient translucent medium
where midge larvae can be seen clearly (particularly when illuminated
from the sides of the Petri dishes instead of from the top) feeding on the
nematodes (late instars more so than early instars). The late instars tend
to engulf the nematodes whole while the early instars on occasion were
found to nibble on nematode pieces. The encounters between midge
larvae and nematodes appeared to be random. Agar concentration ap-
pears to be an important factor in the success of this method. The agar,
if too dense (higher concentrations), does not allow midge larvae to
burrow freely and the larvae end up crawling up the walls of Petri
dishes and die as a result of desiccation. On the other hand, the agar
if too watery (lower concentrations) can result in unfavorable condi-
tions for larvae with the formation of biolm on the surface. We found
that agar concentration of 0.25% provides a good balance between
the agar being too dense or too watery for midge larvae and lasts long
enough till larval development is complete, provided the agar in Petri
dishes is kept moist with standing water during this time (it is possible
that this suitable agar concentration for midge larval rearing [0.25%]
varies with the brand/type of agar used). Pupation occurs usually on
the surface of the agar where pupae are almost entirely submerged with
the prothoracic respiratory horns exposed. However, pupation can also
occur in the standing water sometimes where C.stellifer pupae oat on
the surface of water. This method of midge larval rearing provides easy
visualization/monitoring of midge larval behavior, feeding, growth,
and development and is also much simpler than the traditional way
used to maintain C.sonorensis colonies (Jones etal. 1969).
The egg stage duration of C.stellifer lasted mostly between 3 and
4 d.However, a few eggs on occasion hatched later (up to 7 d; Table
1). The eggs of C.stellifer were also small-sized and banana-shaped,
white in color when laid but turn dark brown over time, typical of
this genus (Mullen and Murphree 2019).
Egg hatch rates ranged from 25.0 to 71.0% and varied signif-
icantly across the study (LR χ
2
3= 61.9, P< 0.0001; Table 1). It is
currently uncertain why there was a signicant variation found in
the egg hatch rates in this study. However, it is possible that this
variation is due to variation in the mated status of males the fe-
males mated with in the eld and/or age of these eld-collected fe-
males that may have inuenced fertilization of the eggs (Degner and
Harrington 2016). Previously, females that mated with prior-mated
males showed lower rates of fertilization than others in Anastrepha
obliqua Macquart (Diptera: Tephritidae) tephritid ies and in
Pararge aegeria Linnaeus (Lepidoptera: Nymphalidae) butteries
(Lauwers and Van Dyck 2006, Perez-Staples etal. 2008).
Complete development of C. stellifer larvae to the adult stage
occurred only in two treatments: 1) nematodes and 2) nematodes
+ LY (Table 1). Culicoides stellifer rst-instar larvae could not sur-
vive beyond 1wk on a diet of microbes alone or in the sterile water
treatment. Larval survival rate to pupal stage (mean ± SE) in the
nematode fed group was 79.2 ± 11.3% (range 61.0–100.0%) but
varied signicantly between the trials (LR χ
2
2 =7.9, P = 0.0189;
Fig. 2A). Mean larval survival rate to pupal stage in the nematode
+ LY group was comparatively slightly lower: 66.5± 19.6% (range
28.8–94.4%), which also varied signicantly between the trials (LR
χ
2
2=45.4, P<0.0001; Fig. 2A). Larval stage duration in the nema-
tode fed group was 21.7± 2.6 d (range 16.9–25.8 d) but varied sig-
nicantly between trials (LR χ
2
2=37.2, P<0.0001; Fig. 2B). Larval
stage duration in the nematode + LY group was not very different
from that in the nematode group: 21.1± 2.4 d (range 16.7–25.0 d),
but also varied signicantly between the trials (LR χ
2
2 = 37.3,
P<0.0001). Larval survival rates were found to be affected by signi-
cant interactions between larval stage duration and larval densities in
the nematode fed group (LR χ
2
1=18.1, P=0.0005) as well as in the
nematode + LY group (LR χ
2
1=10.2, P=0.0014). The frequency dis-
tributions of overall pupation rates were typically skewed to the right
in the nematode group as well as in the nematode + LY group (Fig.
2C). The huge variation observed in larval survival rates and larval
stage durations in the two nematode-fed groups was not unexpected.
It is possible that this variation arose due to variation in the age and
nutritional statuses of the eld-collected females from which eggs
were acquired from and/or due to variation in the number of eggs in
the dishes that varied from 10 to 25 (Table 1), potentially resulting in
variation in larval densities ultimately affecting larval survival/devel-
opment. Previously, insect larval survival and/or development were
shown to be inuenced by genetic factors such as maternal age and
maternal/paternal nutrition (Mousseau and Dingle 1991, Zirbel and
Alto 2018), as well as environmental factors such as larval nutrition,
larval rearing temperatures, and larval densities (Linley 1969, 1985;
Akey etal. 1978; Alto etal. 2012). Alternately, the age/condition of
the nematodes/medium used in this study could also have inuenced
the larval life history traits of C.stellifer. Panagrellus redivivus nema-
todes are sensitive to the timing of food replacement. The popula-
tion of nematodes, particularly the immature stages, explodes as soon
as they are introduced to a fresh batch of medium. However, more
Table 1. Summary of the larval rearing experiments, egg stage duration, and egg hatch rates (mean ± SE) of C.stellifer
Larval diet Trial
(no. of replicates)
No. of eggs/dish Egg stage duration
(d)
Egg hatch rate*
(%)
Complete larval
development
occurred?
Nematodes 1 (3) 20– 21 3–4 57.3± 6.2 Yes
2 (5) 14–20 3–4 25.0± 13.5 Yes
3 (4) 10 3–4 42.5± 13.8 Yes
Nematodes + LY 1 (4) 20–25 3–4 71.0± 6.1 Yes
2 (4) 10 3–4 67.5± 16.0 Yes
3 (4) 10 3–4 85.0± 5.0 Yes
Microbes from nematode medium 1 (3) 10 3–4 50.0± 11.5 No
2 (3) 10 3–4 30.0± 5.8 No
3 (4) 10 3–4 57.5± 12.5 No
Tap water (autoclaved) 1 (3) 11–12 3–4 42.2± 8.0 No
2 (3) 15 3–4 31.1± 5.9 No
3 (3) 15 3–4 36.4± 5.2 No
Asterisk indicates signicant variation in egg hatch rates across the study.
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adults prevail in the medium as time progresses. It is likely that the
early instars of C.stellifer have difculty in capturing/ingesting adult
nematodes, and it is possible that C.stellifer early instars in our study
(at least during some trials) may have been given nematodes that
were mainly adults. Unfortunately, we currently do not have the re-
cords of nematode colony maintenance because the nematodes used
in this study were purchased commercially. Further studies will be
needed to test these hypotheses.
Pupal stage duration was 2.8± 0.3 d (range 2–5 d) in the nem-
atode fed group but was longer in the nematode + LY group (4.2±
0.3 d, range 3–6 d; Fig. 2D). There were no signicant differences
in pupal stage duration between trials in the nematode fed group
(LR χ
2
2= 0.6, P =0.7361) as well as in the nematode + LY group
(LR χ
2
2=0.4, P=0.8292). It is possible that nutritional differences
between the two treatments inuenced pupal stage duration in
C.stellifer. Previously, duration of various insect life stages including
that of the pupal stage has been shown to vary with different larval
diets (Zhang etal. 2007, Puggioli etal. 2013).
Adult eclosion rates from the pupal stage, in general, were high
(≥94.7%) and were not signicantly different between trials in the
nematode-fed group (LR χ
2
2=1.4, P=0.4896) as well as in the nem-
atode + LY group (LR χ
2
2=1.2, P=0.5528; Fig. 3A). Sex ratios of
the F1 adults were ~1:1 (M:F) in the nematode-fed group and not
signicantly different between trials (LR χ
2
2=0.0, P=0.9790; Fig.
3B), whereas sex-ratios, overall, were male biased in the nematode
+ LY group (~2:1) and were not signicantly different between the
trials (LR χ
2
2=2.6, P=0.2719; Fig. 3B).
The overall development of C.stellifer using the nematode diet
was found to be satisfactory for potential colonization purposes as
it resulted in high-larval survival rates and nondistorted sex-ratio
in the F1 adults (~1:1) of this species. Interestingly, previous studies
using the agar and nematode method resulted in nondistorted F1
sex-ratios only in C. circumscriptus (Barceló and Miranda 2018),
but caused male-biased F1 sex-ratios in C.imicola (Veronesi et al.
2009, Barceló and Miranda 2018), C. obsoletus (Boorman 1985,
Barceló and Miranda 2018), C. aterinervis (Kitaoka 1982), and
C.newsteadi (Barceló and Miranda 2018), and female-biased sex-
ratios in C. matsuzawai (Kitaoka 1982), C. cataneii (Barceló and
Miranda 2018), and C. paolae (Barceló and Miranda 2018), sug-
gesting that nematode diet may not be ideal for laboratory rearing
of all Culicoides species. Furthermore, these ndings collectively
suggest that that nutritional requirements of larvae vary between
Culicoides species. Indeed, based on feeding habits and the asso-
ciated mouthparts, Culicoides larvae have been broadly classied
into two groups: 1)herbivorous larvae that feed on detritus, bac-
teria, fungi, or other organic material and 2) carnivorous larvae
Fig. 2. Larval survival rates (mean ± SE) (A) and larval stage duration (B) of C.stellifer reared on a diet of nematodes or nematodes + lactalbumin and yeast (LY).
Letters above bars indicate significant differences between trials within each treatment (P<0.05). Frequency distributions of the overall pupation rates (three
trials pooled) of C.stellifer reared on different diets (C). Overall pupal stage duration of C.stellifer reared on different diets (D). Culicoides stellifer larvae could
not survive beyond one week on a diet of microbes alone or in the sterile water treatment.
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(predaceous) that feed on protozoans, oligochaetes, nematodes, or
other invertebrates (Hribar and Mullen 1991, Hribar 1993, Mullen
and Murphree 2019). However, Culicoides larvae are evidently om-
nivorous opportunistic feeders (Aussel and Linley 1994, Mullen and
Murphree 2019). Aclassic example being larvae of C. sonorensis
that can grow on a diet of microbes (bacteria and fungi; Jones etal.
1969, Parker etal. 1977), but can also grow when fed nematodes
(Mullens and Velten 1994). Unfortunately, nothing is known re-
garding the feeding habits of C. stellifer larvae, anatomy of their
mouthparts, or their nutritional requirements currently. Further
studies will be needed to elucidate these fundamental aspects of
C.stellifer and other species in general as larval nutritional require-
ments are among the least understood aspects of Culicoides spe-
cies to date. Regardless, our current ndings suggest that the agar
and nematode method can be successfully used to rear C. stellifer
larvae under laboratory conditions. However, further studies will be
needed to examine larval development of C.stellifer on other species
of nematodes or other potential diets/supplements that could help
optimize laboratory rearing conditions of this midge species (Linley
1985).
It was interesting that nematodes supplemented with LY re-
sulted in slightly lower larval survival rates on average than the
diet of nematodes alone. Moreover, sex-ratio of the F1 adults was
male-biased in the nematode + LY treatment but were nondistorted
(~1:1) in the nematode-fed group. Currently, reasons behind these
outcomes are unknown. However, it is possible that the addition of
LY to agar + nematode medium resulted in the substrates becoming
too eutrophic and caused an overgrowth of microbes and ultimately
increased female larval mortality by reducing their nutrient availa-
bility. Previously, female mosquito larvae have been suggested to re-
quire greater nutritional resources to pupate than males (Chambers
and Klowden 1990, Zirbel etal. 2018). Alternately, the addition of
LY to the agar + nematode medium may have altered certain en-
vironmental conditions in the medium that increased larval midge
mortality, particularly in females. Indeed, agar in the nematodes + LY
treatment was visibly discolored within 2wk and resulted in thick
slurry formation on the agar/water surface, which was possibly un-
favorable for female larval survival. However, further studies will be
needed to test these hypotheses.
A live microbial community in the substrate functions as a di-
rect source of nutrition to insect larvae by serving as food (Terra
and Ferreira 1994), or as an indirect source of nutrition by breaking
down the substrate for larval digestion/absorption, synthesizing vi-
tamin B supplements for insect larvae (Hobson 1932a, 1932b, 1933),
or by even providing signaling cues/conditions that regulate growth
and development in insects (Shin et al. 2011, Storelli et al. 2011,
Coon etal. 2017). In conjunction with these reports, previous studies
have shown that various insect larvae including Culicoides species
fail to develop in sterile environments, and that larval development
is rescued when sterile substrates are inoculated with live microbes
(Kettle etal. 1975, Williams and Turner 1976, Vaughan and Turner
1987, Zurek etal. 2000, Romero etal. 2006, Peterkova-Koci et al.
2012). Consistent with previous reports, C.stellifer larvae, in our
study, could not develop under sterile conditions, suggesting that
microbial community is required for midge larval survival/develop-
ment. However, C.stellifer larvae could not survive beyond 1wk on
a diet of microbes, with larval mortality beginning early on (day 2
onwards), suggesting that a microbial dietalone does not meet all
nutritional requirements of midge larvae (even early instars). It is
likely that microbe only diet does not satisfy protein, lipid, or other
fatty acid requirements of C. stellifer larvae. Previously, various
amino acids and lipids including sterols were found to be essential
for mosquito larval development (Golberg and De Meillon 1948a,
1948b; Singh and Brown 1957; Dadd and Kleinjan 1984; Merritt
et al. 1992). Nematode diet, on the other hand, likely satises all
nutritional requirements of C. stellifer larvae and was reected in
the generally high larval survival rates in the nematode fed groups
(Schlechtriem etal. 2004a, 2004b). Previously, larvae of C.melleus
were also found to not survive on a diet of bacteria alone, but devel-
oped well when fed nematodes (Linley 1985).
Mating in Culicoides species is believed to occur typically
in ight. Males form swarms (usually in response to host cues,
larval habitat cues, vegetation cues, or dawn/dusk cues) that the
females y into and are captured by males for copulation (oblig-
atory swarmers [eurygamy]; Downes 1955). However, some spe-
cies can mate in restricted spaces and do not require swarming to
mate (facultative swarmers [stenogamy]; Linley and Adams 1972,
Roberts et al. 1977, Jones and Schmidtmann 1980, Gerry and
Mullens 1998, Mair and Blackwell 1998). Currently, very little is
known regarding the factors/cues involved in swarming/mating in
biting midges (Linley and Adams 1972, Campbell and Kettle 1979,
Gerry and Mullens 1998, Mair and Blackwell 1998, González etal.
Fig. 3. Overall adult eclosion rates (three trials pooled [mean ± 95% CI]) of C.stellifer reared on a diet of nematodes or nematodes + lactalbumin and yeast (LY)
(A). Overall sex-ratios of C.stellifer F1 adults reared on different diets (B). Culicoides stellifer larvae could not survive beyond 1wk on a diet of microbes alone
or in the sterile water treatment.
5Journal of Medical Entomology, 2019, Vol. 00, No. 00
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2017, Kirkeby 2018), because of which only few Culicoides species
have been successfully colonized to date (Linley 1968, Roberts et
al. 1977, Jones et al. 1969, Sun 1969, Boorman 1974, Williams
and Turner 1976, Koch and Axtell 1978, Jones and Schmidtmann
1980, Mullens and Schmidtmann 1981). Our attempts to induce
mating in the F1 adults of C.stellifer by providing olfactory cues
from the host (octenol), habitat (mud + cattle manure), vegetation
(Sphagnum spp. moss), or dawn/dusk conditions have been futile.
Furthermore, holding the F1 adults in 47.5× 47.5× 47.5cm cages
(BugDorm-4F4545), 1.9-liter paper cups, 500.0-ml paper cups, as-
pirator tubes (1.0cm diameter), or in capillary tubes (with male
and female terminalia in contact) also did not induce swarming/
mating in this species (nonmating was deduced based on no depo-
sition of viable eggs by the F1 females post bloodmeal). The mating
behavior of C.stellifer has never been documented before; there-
fore, it is currently uncertain whether increased/decreased cage
space, or cues from certain host animals, vegetation, or a certain
combination of cues including visual/tactile cues may help accom-
plish mating in this species under laboratory conditions. Further
studies will be needed to examine the reproductive behavior of
C. stellifer under eld conditions, ascertain whether this species
is an obligatory or facultative swarmer, and identify the potential
mating/swarming cues utilized by this species. This information
could be useful in designing cages and/or providing conditions
that encourage mating of C.stellifer adults under laboratory con-
ditions. In addition, further studies will also be needed to examine
whether other alternatives, such as red or blue lights (Baerg 1971),
light beams from ash light in combination with lowered temperat-
ures (Villarreal etal. 1998), stroboscopic blue lights (Lardeux etal.
2007), or forced mating (McDaniel and Horsfall 1957, Baker etal.
1962, Sun 1969, Amir etal. 2013) could be used to induce captive
mating in C.stellifer and other important species associated with
Orbivirus transmission in North America.
Conclusions
Our ndings collectively suggest that microbial community is re-
quired for the larval development of C.stellifer. However, microbial
community alone is not sufcient, other nutritional components such
as proteins or lipids may also be essential to support midge larval
development. Further studies will be needed to examine the larval
nutritional requirements of C.stellifer and other important species.
Overall, the agar and nematode method can be successfully used to
rear C. stellifer larvae under laboratory conditions. However, fur-
ther studies will be needed to examine the development of C.stellifer
larvae on other potential diets/supplements, which could help op-
timize laboratory rearing conditions of this species. Unfortunately,
captive mating of F1 individuals remains a major obstacle towards
successful colonization of this species currently. Further studies will
be needed to examine the mating behavior of C.stellifer under eld
conditions and identify the potential mating/swarming cues utilized
by this species. This information could be useful in providing con-
ditions that encourage mating of C.stellifer F1 adults under labora-
tory conditions.
Acknowledgments
We thank Alfred Runkel, Bethany McGregor, Kristin Sloyer, and
Erik Blosser for assisting with the collection and/or maintenance of
live eld-collected midge adults. Agustin Quaglia provided help with
statistical analysis of the data and reviewed an earlier version of this
manuscript. We also thank the commercial deer farmers in Quincy,
FL, USA for allowing us to collect live midges from their property.
Funding for this study was provided by the University of Florida,
Cervidae Health Research Initiative (CHeRI) sponsored by the State
of Florida legislature.
Author Contributions
Dinesh Erram: Conceptualization, Methodology, Data Curation,
Formal Analysis, Visualization, Validation, Investigation, Resources,
Project Administration, Writing – Original Draft Preparation.
Nathan Burkett-Cadena: Funding Acquisition, Supervision, Writing
– Review & Editing.
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