Medical and Veterinary Entomology (2014), doi: 10.1111/mve.12086
Sugar-feeding behaviour and longevity of European Culicoides biting midges C. K A U F M A N N 1 , A. M A T H I S 1 and C. V O R B U R G E R 2 1
National Centre for Vector Entomology, Institute of Parasitology, Vetsuisse Faculty, University of Zürich, Zürich, Switzerland and Institute of Integrative Biology, ETH Zürich and EAWAG (Swiss Federal Institute of Aquatic Science and Technology), Dübendorf, Switzerland 2
Abstract. Most haematophagous insect vectors can also use sugar as an energy source; thus their sugar-feeding behaviour influences their longevity and blood-feeding rate and hence their vectorial capacity. Scant information is available on the sugar-feeding behaviour of Culicoides Latreille biting midges (Diptera: Ceratopogonidae), which are vectors of bluetongue and Schmallenberg viruses. The longevity of laboratory-reared Culicoides nubeculosus (Meigen) under fluctuating temperatures (16 and 28 ∘ C) and with access to water or water and blood was on average 6.4 days and 8.9 days, respectively, which was around one third of the lifespan of siblings with access to sugar or sugar and blood (22.2 days and 27.1 days, respectively). Access to honeydew significantly increased the midge’s longevity, whereas the provision of extrafloral nectaries had no impact. Females with access to sugar produced a significantly higher number of eggs (65.5 ± 5.2) than their starved sisters (45.4 ± 8.4). More than 80% of field-caught female Culicoides from the two most abundant European groups, Obsoletus (n = 2243) and Pulicaris (n = 805), were fructose-positive. Fructose-positivity was high in all physiological stages and no seasonal variability was noted. The high rate of natural sugar feeding of Culicoides offers opportunities for the development of novel control strategies using toxic sugar baits and for the monitoring of vector-borne diseases using sugar-treated FTA (nucleic acid preservation) cards in the field. Key words. Aphids, blood, control strategies, fructose, honeydew, nectar, vectors.
Introduction Most biting flies rely on sugar sources in order to supplement their blood diet with carbohydrates. Naturally occurring sugar sources exploited by biting flies include floral and extrafloral nectaries, and fruits (damaged, rotten or dried fruits), as well as honeydew (Downes, 1958; Foster, 1995; Engel et al., 2001; Stanfield & Hunter, 2010; Muller et al., 2012). In addition, mosquitoes (Diptera: Culicidae) and sandflies (Diptera: Psychodidae) have been shown to access nutritious plant sap directly from vegetative tissues (Schlein & Jacobson, 1994; Schlein & Muller, 1995; Junnila et al., 2010). Sugar is used as a nutritional source that is additional to teneral energy reserves and can be stored as glycogen or lipid (Van Handel, 1965). This ‘sweet
source’ improves the insect’s flight performance (e.g. Kaufmann & Briegel, 2004), as well as mating rates and overall reproductive success (Gary et al., 2009; Gu et al., 2011; Stone et al., 2011). Sugar feeding also contributes to the survival of diapausing adults (Mitchell & Briegel, 1989) and to longevity in general (Jamnback, 1961; Gary & Foster, 2004; Impoinvil et al., 2004). The sugar-feeding behaviour of biting flies has been studied thoroughly, particularly in mosquitoes (reviewed in Foster, 1995), and has revealed distinct characteristics. The Asian tiger mosquito Aedes albopictus (Stegomyia albopicta) Skuse (Diptera: Culicidae) relies on sugar intake as its mean longevity without sugar stops is only a few days (Xue et al., 2010). By contrast, sugar feeding was observed to be rare in the yellow fever mosquito, Aedes aegypti (L.) (Stegomyia aegypti) (Edman
Correspondence: Alexander Mathis, National Centre for Vector Entomology, Institute of Parasitology, Vetsuisse Faculty, University of Zürich, Winterthurerstrasse 266a, CH-8057 Zürich, Switzerland. Tel.: + 41 44 635 85 36; Fax: + 41 44 635 89 07; E-mail: [email protected] © 2014 The Royal Entomological Society
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2 C. Kaufmann et al. et al., 1992), and similar observations were reported for the African malaria mosquitoes Anopheles gambiae Giles s.l. and Anopheles funestus Giles (Diptera: Culicidae) (Beier, 1996). However, sugar intake in these mosquitoes varies depending on season and natural habitat, and can be considerable (Bidlingmayer & Hem, 1973; Spencer et al., 2005; Gu et al., 2011). The European malaria vector Anopheles atroparvus Thiel is dependent on sugar sources to achieve strong flight performance, whereas the main African malaria vector, An. gambiae, reaches comparable flight distances when fed either only with sugar or only with blood (Kaufmann & Briegel, 2004). Sugar uptake can boost the vectorial capacity of mosquitoes by enhancing their longevity beyond the extrinsic incubation period of the disease agents they transmit (Xue et al., 2010). However, the biting activity of the main African malaria vector An. gambiae can increase if sugar is lacking (Gary & Foster, 2001), resulting in the earlier transmission of vector-borne disease. Clearly, knowledge about the sugar-feeding behaviour of vector species is an important component in assessments of their vectorial capacity. A few studies of the natural sugar-feeding behaviour of Culicoides biting midges, which are vectors of the bluetongue and Schmallenberg viruses, have been carried out in the U.S.A. [i.e. in Culicoides melleus (Coquillet) and Culicoides hollensis (Melander and Brues) (Magnarelli, 1981), Culicoides variipennis (Coquillet) (Mullens, 1985), and Culicoides mississippiensis Hoffman (Stewart & Kline, 1999)]. Further, wild-caught Culicoides obsoletus (Meigen) were shown to live longer in the laboratory when supplied with sugar (Jamnback, 1961). The data available on the main incriminated European vectors (i.e. species of the Obsoletus and Pulicaris groups) are scant with regard to their general biology, including their sugar-feeding behaviour (Carpenter et al., 2009). Species of the Obsoletus group are known to have breeding habitats in the close surroundings of livestock farms (Kettle & Lawson, 1952; Zimmer et al., 2008). Hence, suitable blood hosts can be found in close proximity to eclosed midges, which raises the question of whether midges rely on natural sugar sources. In the present study, the sugar-feeding behaviour of Culicoides spp. from the Obsoletus and Pulicaris groups, caught on a farm in Switzerland, was assessed. In addition, possible sugar ingestion from different sources (floral and extrafloral nectaries, honeydew and sugar solution) and its effects on longevity and reproduction were investigated using Culicoides nubeculosus (Meigen) as a laboratory model organism.
Materials and methods Insects and climatic conditions The experimental work was carried out with laboratory-reared C. nubeculosus ceratopogonid biting midges, originally kindly provided by Dr S. Carpenter, Pirbright Institute, Woking, U.K. Insects were reared as described in Boorman (1974), except that larvae were fed with pulverized Tetramin® only and blood feeding was achieved through a Nescofilm® membrane with heparinized sheep blood. In the main colony, insects were kept at a mean ± standard deviation (SD) temperature of 24 ± 0.5 ∘ C and
relative humidity (RH) of 85 ± 5% under long day conditions (LD 16 : 8 h) that included dawn and dusk periods of 1 h each. All experiments were performed in a climate chamber (Kälte 3000 AG, Landquart, Switzerland) under summer day conditions (i.e. temperature range: 16–28 ∘ C; mean ± SD temperature: 22 ± 0.5 ∘ C; RH: 60–90%; mean ± SD RH: 78 ± 5%) (Fig. 1) and a long-day setting as described above. The minimum RH of 60% was set to occur at the early afternoon peak in temperature. Indigenous biting midges were caught alive on a livestock (i.e. cows, horses and chickens) farm in Switzerland (47∘ 22′ 30.86′′ N, 08∘ 34′ 57.86′′ E). A modified Onderstepoort ultraviolet (UV) light suction trap was used for trapping, as described previously (Venter & Meiswinkel, 1994), and a cubic cage (24.5 × 24.5 × 24.5 cm3 ; BugDorm-42222F; MegaView Science Co., Ltd, Taichung, Taiwan) was used as a trapping vessel. Trapping was carried out overnight (including dawn and dusk) in spring (April and May), summer (June and August) and autumn (late September). After trapping, cages were placed in thermally insulated containers chilled with ice and transported to the nearby institute where the insects were killed at −20 ∘ C and then sorted into Obsoletus and Pulicaris group specimens. Although no attempts were made to identify the investigated Culicoides to species level, the most likely species at this site, as deduced from the identification by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry of midges collected from comparable sites (Kaufmann et al., 2012) were C. obsoletus, Culicoides scoticus Downes and Kettle (Obsoletus group) and Culicoides pulicaris (L.), Culicoides lupicaris Downes and Kettle, and Culicoides grisescens Edwards (Pulicaris group). Females were additionally divided into the physiological stages of nulliparous, parous, gravid and blood-fed. If they were not to be immediately processed for fructose analysis, insect samples were stored at −80 ∘ C.
Longevity with experimental diets Longevity experiments were performed in cubic cages (17.5 × 17.5 × 17.5 cm3 ; BugDorm-41515) under five different diets: (a) no nutrition; (b) water; (c) 10% sucrose solution; (d) water and blood, and (e) 10% sucrose solution and blood. Water and sucrose solutions were available ad libitum; heparinized sheep blood was provided daily through a Nescofilm® membrane for 30 min. The experiments were repeated in triplicate. To start the experiment, 10 newly eclosed C. nubeculosus of each sex were transferred to each experimental cage. Survival was monitored daily; dead individuals were removed and their sex determined. Every second day, eggs laid on a wet filter paper in the cage were removed and discarded.
Longevity and sugar ingestion with natural sugar sources Two experiments were carried out to investigate the midges’ capability to use naturally occurring sugar sources and their effect on longevity. The first experiment comprised four treatments. All cages (BugDorm-42222F) were supplied with a
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12086
Sugar feeding of Culicoides biting midges
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Fig. 1. Temporal temperature ( ) and humidity ( ) profiles for simulated summer days.
non-flowering broad bean plant (Vicia faba), a vetch producing extrafloral nectaries on leaf stipules (Engel et al., 2001), on which the presence of extrafloral nectaries was manipulated (left intact or stipules removed with forceps), with or without the presence of honeydew-producing black bean aphids (Aphis fabae Scopoli) (Hemiptera: Aphidae) in a 2 × 2 factorial design. The presence and absence of aphids (providing honeydew) and/or of nectar in extrafloral nectaries were visually confirmed. Each treatment was replicated three times. A first set of 10 male and 10 female C. nubeculosus was added to the cages for 1 day, then removed and analysed for fructose ingestion using the cold anthrone test (see below). A second set of midges was then added to these cages (with permanent access to the different natural sugar sources) to monitor longevity as described under ‘Longevity with experimental diets’ above. In the second experiment, midges were given access to two spring flowering plants (the daisy, Bellis perennis and the lesser celandine, Ranunculus ficaria) for 1 day. Control cages contained neither food nor water. The daisy treatment was replicated in four cages, the lesser celandine treatment in three cages and the control treatment in two cages.
Fecundity Female and male C. nubeculosus were maintained in cubic cages (BugDorm-41515) with permanent access to water only or to 10% sucrose. One single bloodmeal (heparinized sheep blood) was given when females were 3 days old; only females that were visually determined using a stereomicroscope to be fully engorged were included in the further trial. After an additional 3 days of incubation time, females were dissected in order to count their eggs (n = 24 females for each treatment).
pellet pestles (Fisher Scientific AG, Wohlen, Switzerland). Each cohort of samples tested included two negative controls and a standard of 0.1% fructose (Sigma-Aldrich Chemie GmbH) in ethanol (25%). A preliminary experiment was performed using the in-house C. nubeculosus colony. Twenty-five insects with access to water only were all found to be negative in the cold anthrone test, whereas 96% of insects with access to 10% fructose solution for 1 h (after being deprived of water for 6 h) were positive (visual observation of colour changes from yellowish to green).
Statistical analysis Survival data were analysed with Cox proportional hazards models using the open source statistical software R Version 2.13.0 (R Development Core Team, 2011), testing for the effects of sex and the different treatments on survival. To account for the non-independence of midges developing in the same cages, we used the coxme library, a contributory package to R for Cox mixed-effects models, which allowed us to enter ‘cage’ as a random effect in our models. The data on sugar ingestion in the experiments were treated as binomial (fructose-negative or fructose-positive) and analysed using generalized linear mixed models (GLMMs) with the logit link and binomial errors, executed with the lme4 library in R. Again, the effects of sex were tested for in the different treatments and ‘cage’ was included as a random effect to account for the non-independence of biting midges kept in the same cage. Tukey contrasts were used for multiple comparisons of means as implemented in the multcomp library (Hothorn et al., 2008).
Results Fructose-sensitive cold anthrone test
Longevity with experimental diets
The presence of fructose was determined using the cold anthrone test according to Van Handel (1972). In brief, whole insects were homogenized in 0.2 mL of anthrone reagent (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) using 1.5-mL microtubes with a hand-homogenizer and disposable
Culicoides nubeculosus were kept under five different diets (no food, water, sucrose solution, and blood feeding with access to water or sucrose), and all experiments were repeated three times. Overall, mortality was higher in males than in females in this experiment (Z = 2.36, P = 0.018). Although there was
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12086
4 C. Kaufmann et al. Table 1. Longevity of Culicoides nubeculosus biting midges∗ under climatic summer day conditions† and different experimental diets (water, sucrose, blood). Longevity, days
Longevity with natural sugar sources
Diet
Sex
Mean
Median
Maximum
NF
♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂
4.27 3.70 6.40 5.27 8.87 5.63 22.17 17.43 27.07 16.20
4.00 4.00 6.50 5.00 8.00 5.00 18.00 15.00 27.50 15.00
5 5 8 6 13 8 51 30 40 28
WF WBF SF SBF
to a positive effect on female survival (Table 1, Fig. 2) because males do not feed on blood.
∗n = 10 males and 10 females per treatment, repeated in triplicates. †16–28 ∘ C, 60–90% relative humidity, light regimen LD 16 : 8 h. NF, no food; WF, water-fed; WBF, water and blood-fed; SF, sucrose-fed; SBF, sucrose and blood-fed. Water and 10% sucrose solution were available ad libitum; blood-feeding through an artificial membrane was offered daily for 30 min.
significant variation in mortality rates among cages within treatments (accounted for by the random cage effect in the model: 𝜒 2 1 = 13.88, P < 0.001), clear differences among food treatments on mortality were evident. Midges with access to sucrose in their diet lived considerably longer than those with no access to sucrose (Table 1, Fig. 2), independent of the additional availability of blood (sucrose-fed vs. sucrose and blood-fed treatment: Z = −0.104, P = 0.999). Mortality was highest in the no-food (NF) treatment (Table 1, Fig. 2). Access to water only (WF) decreased mortality significantly (NF vs. WF: Z = −6.22, P < 0.001), and access to blood in addition to water (WBF treatment) decreased mortality further (WF vs. WBF: Z = 3.38, P = 0.006). Not surprisingly, this effect was mainly attributable
In the first experiment with natural sugar sources (extrafloral nectaries; honeydew produced by black bean aphids), C. nubeculosus males died earlier than females (Z = 3.32, P < 0.001) but there was no significant variation in mortality rates among cages within treatments (𝜒 2 1 = 0.46, P = 0.498). Inspection of longevities and survivorship curves (Table 2, Fig. 3) clearly shows that the presence of aphids and thus access to their excreted honeydew strongly reduced mortality (Z = −6.23, P < 0.001), whereas the presence of extrafloral nectaries did not significantly improve survival (Z = −1.51, P = 0.130). There was no significant interaction between the presence of aphids and extrafloral nectaries on midge mortality (Z = 1.47, P = 0.140).
Sugar-feeding behaviour in the laboratory and field Fructose analysis with the cold anthrone test confirmed that C. nubeculosus ingested aphid honeydew but very little nectar from plant extrafloral sources. Thus, in the presence of aphids, 68–89% of midges ingested sugar (Fig. 4), but only 3–17% were fructose-positive in the absence of aphids. Accordingly, aphid presence was the only significant effect in the model (Z = 3.24, P = 0.001) and the presence of extrafloral nectaries had no significant effect (Z = 0.63, P = 0.532). Males overall tested positive for fructose more frequently than females, but this difference was not statistically significant (Z = 1.59, P = 0.113). Two common blooming flowers (the daisy and lesser celandine) were also tested as potential natural sugar sources for C. nubeculosus and were found to differ markedly in their suitability. Only a few (7% of males) of the laboratory biting
Fig. 2. Longevity of male and female Culicoides nubeculosus biting midges kept under summer day (16–28 ∘ C, 60–90% relative humidity, light regimen LD 16 : 8 h) climatic conditions on different diets. , no food; , water-fed; , water and blood-fed; , sucrose-fed; , sucrose and blood-fed. © 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12086
Sugar feeding of Culicoides biting midges
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Table 2. Longevity of Culicoides nubeculosus biting midges∗ under climatic summer day conditions†and different sources (natural sugars). Longevity, days Source
Sex
Mean
Median
Maximum
N− /A−
♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂
6.67 5.62 7.11 5.90 13.36 8.37 12.60 9.73
7.00 5.00 7.00 6.00 14.50 8.00 12.00 9.50
8 7 10 9 20 16 18 14
N+ /A− N− /A+ N+ /A+
∗n = 10 males and 10 females per treatment, repeated in triplicates. †16–28 ∘ C, 60–90% relative humidity, light regimen LD 16 : 8 h. N− /N+ : without or with access to extrafloral nectaries on field bean plants Vicia faba; A− /A+ : without or with infestation with the black bean aphid Aphis fabae producing honeydew.
midges accessed the nectar of daisies, whereas 58% of tested females and 67% of tested males accessed the lesser celandine nectar (Fig. 4). All control midges with access to water only were fructose-negative (Fig. 4). The GLMM to compare sugar ingestion between midges with access to daisies and lesser celandine flowers, respectively, did not converge because no females from daisy cages tested positive. To make the model converge and obtain a conservative test for the difference between flowers, we artificially set one female from a daisy cage as fructose-positive. This conservative comparison still showed a highly significant difference (Z = 3.61, P < 0.001), whereas the difference in sugar ingestion between males and females was not statistically significant (Z = 0.78, P = 0.437). At a farm, we collected 2243 Culicoides of the Obsoletus group (2169 females, 74 males) and a total of 805 specimens of the Pulicaris group (796 females, 9 males), all of which
Fig. 4. Percentages of fructose-positive male (black bars) and female (white bars) Culicoides nubeculosus biting midges after 24 h of access to the broad bean plant Vicia faba without (N− ) or with (N+ ) extrafloral nectaries and without (A− ) the black bean aphid Aphis fabae (N− /A− ; N+ /A− ) or with access to V. faba (without or with extrafloral nectaries) infested with (A+ ) A. fabae (N− /A+ ; N+ /A+ ). In addition, both sexes of C. nubeculosus were tested with access to daisies (D), Bellis perennis, lesser celandines (P), Ranunculus ficaria, or under non-feeding conditions (n = 10 males/females, repeated in triplicates; climatic summer day conditions: 16–28 ∘ C, 60–90% relative humidity, LD 16 : 8 h).
were individually subjected to the anthrone test. In both groups, the percentage of fructose-positive individuals was higher in females than in males [Obsoletus: 83% vs. 66% (𝜒 2 1 = 12.77, P < 0.001); Pulicaris: 80% vs. 33% (𝜒 2 1 = 9.213, P = 0.002)] (Table 3), but the figures for males were obtained from much lower numbers of individuals. Sugar-feeding behaviour also differed among the physiological stages of females in both groups (Obsoletus: 𝜒 2 3 = 51.50, P < 0.001; Pulicaris: 𝜒 2 3 = 9.91,
Fig. 3. Longevity of male and female Culicoides nubeculosus biting midges kept at summer day (16–28 ∘ C, 60–90% relative humidity, light regimen LD 16 : 8 h) climatic conditions with permanent access to the field bean plant Vicia faba with (N+ ) or without (N− ) extrafloral nectaries and with (A+ ) or without (A− ) infestation with the black bean aphid Aphis fabae. , N− /A− ; , N+ /A− ; , N− /A+ ; , N+ /A+ . © 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12086
6 C. Kaufmann et al. Table 3. Percentages of fructose-positive Culicoides spp. caught at a farm site in Switzerland. Anthrone-positive samples, % (total n investigated) Stage Group
Sex
Nulliparous Parous Gravid Blood-fed ♀
Obsoletus 86 (1348) Pulicaris 83 (506)
83 (442) 77 (270)
70 (371) 59 (17)
88 (8) 100 (3)
♂
83 (2169) 80 (796)
66 (74) 33 (9)
Table 4. Seasonal prevalences of fructose-positive female Culicoides spp. caught at a farm site in Switzerland. Anthrone-positive samples, % (total n investigated) Group
Spring
Summer
Autumn
Obsoletus
88 (4410) 83 (120)
82 (1539) 81 (614)
82 (189) 73 (62)
Pulicaris
P = 0.019) (Table 3). The percentage of fructose-positive insects was around 80% or more in all stages, with the exception of gravid females (70 and 59% for the Obsoletus and Pulicaris groups, respectively). However, the numbers of gravid (n = 17 in the Pulicaris group) and blood-fed (n = 8 in the Obsoletus group, n = 3 in the Pulicaris group) females were very low. There was little evidence for any seasonality in the sugar-feeding behaviour of Culicoides at this farm site. Although the percentages of fructose-positive females differed significantly among the spring, summer and autumn samples (Obsoletus: 𝜒 2 2 = 8.99, P = 0.011; Pulicaris: 𝜒 2 2 = 16.18, P > 0.001), the values were consistently high throughout the year (Table 4).
Egg production with access to sucrose or under starvation To assess a potential effect of sugar availability on fecundity, a single bloodmeal was provided to midges. Dissection of 24 females per group showed that females with access to 10% fructose contained a significantly higher amount of eggs (65.5 ± 5.2) than their starved siblings (45.4 ± 8.4), which had access to water only (P < 0.001, two-sided t-test).
Discussion The high percentage (around 80% or more) of fructose-positive Culicoides of both Obsoletus and Pulicaris group species, collected at a farm site, was not expected. As the anthrone test used is known to more reliably detect recent sugar feeding (Mullens, 1985), these findings strongly indicate that these Culicoides, which are most abundant around farm sites in Central Europe,
frequently access natural sugar sources. In addition, the high level of sugar feeding among all the different life stages of the field-caught midges (nulliparous, parous, gravid and blood-fed) (Tables 3 and 4) confirms the necessity to access natural sugar sources throughout the lifespan. Seasonal and environmental factors have been shown to influence sugar intake behaviour in mosquitoes (Bidlingmayer & Hem, 1973; Spencer et al., 2005; Gu et al., 2011). However, in the present study, no differences were observed between the seasons evaluated (late spring, summer, autumn). During the cold winter months in Switzerland, there is barely any Culicoides activity (Kaufmann et al., 2009). Levels of fructose-positivity of 66 and 64% were identified in the saltwater marsh species C. melleus and C. hollensis, respectively, when caught with an aspirator upon landing or when blood feeding on humans (note that host blood does not contain fructose) (Magnarelli, 1981). Fructose-positivity of 56% was found in C. mississippiensis collected by sweeping the midges directly from their principal sugar source, the flowering yaupon holly (Ilex vomitoria) (Stewart & Kline, 1999). By contrast, in a study conducted in southern California, only 12% of nulliparous and 24% of parous females of C. variipennis were found to be fructose-positive throughout the year, with the highest percentages occurring between December and May (Mullens, 1985). This suggests rather low sugar consumption, but may be explained by the fact that the midges were caught with carbon dioxide-operated Centers for Disease Control (CDC) miniature traps without light, which mainly attract host-seeking midges. By contrast, the low level of sugar-feeding behaviour in C. variipennis may reflect facultative sugar consumption. A similar strategy was shown in An. gambiae, which survive and fly well on blood alone when natural sugar sources are rare as long as plenty of human hosts are available (McCrae, 1989; Kaufmann & Briegel, 2004), but benefit from additional sugar sources (Gary & Foster, 2001, 2004). The lifespan of Culicoides was substantially increased with access to sugar, as shown for C. nubeculosus in this laboratory study, confirming earlier findings with field-collected C. obsoletus (Jamnback, 1961) and Culicoides brevitarsis (Campbell & Kettle, 1975). Culicoides nubeculosus with access to blood, but not sugar, exhibited short lifespans of about 1 week in our experiments, similar to the observations described for C. obsoletus (Jamnback, 1961). With regard to the transmission of pathogens, a lifespan of 1 week would be sufficient at higher temperatures. The extrinsic incubation period for bluetongue virus in Culicoides sonorensis was shown experimentally to be < 5 days at temperatures of > 20 ∘ C (Carpenter et al., 2011). Thus, virus transmission by Culicoides spp. seems possible without an additional sugar supply. However, in vector competence studies, biting midges have been maintained with permanent access to sugar solution (e.g. Carpenter et al., 2011) and do not need to fly long distances in the laboratory (e.g. to find hosts or breeding sites), which would require additional energy in the field. Our laboratory and field results demonstrated that blood alone is far from optimal for survival and the accumulation of maternal reserves. Hence, we conclude that sugar stops increase the vectorial capacity of biting midges by extending their longevity and the higher potential number of bloodmeals taken during their lifespan.
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12086
Sugar feeding of Culicoides biting midges Interestingly, males of C. nubeculosus showed a slightly higher prevalence of sugar ingestion than females under all experimental conditions (Fig. 4). By contrast, in all field studies, males were consistently less frequently fructose-positive than females (Mullens, 1985; Stewart & Kline, 1999), a finding that held true in this study in field-caught males. Fructose-positivity in males decreased during dusk, which may be related to their mating and flight activity (Stewart & Kline, 1999). Thus, in laboratory cages, reduced flight activity in males may result in lower energy consumption and therefore in a higher probability that fructose-positive insects will be detected. The current study showed that floral nectar and honeydew, which are important sugar sources for mosquitoes (Foster, 1995), sandflies (Molyneux et al., 1991) and blackflies (Stanfield & Hunter, 2010), can be accessed and utilized easily by C. nubeculosus, and result in increased longevity, whereas other sources such as extrafloral nectaries or plant sap were rarely or not accessed. However, we cannot exclude the possibility that such sources are occasionally used by Culicoides, and more potentially relevant plant species should be tested because mosquitoes and sandflies have previously been found to show different preferences in accessing plant tissues from 15 different plant species (Schlein & Muller, 1995). The frequent sugar consumption of field-caught Culicoides spp. may possibly indicate autogenous reproduction for the first batch of eggs, which is known in only a few Culicoides species (summarized in Linley, 1983). However, few data about autogeny are available for species of the Obsoletus and Pulicaris groups. Jamnback (1961) suggested autogeny for C. obsoletus because unmated sugar-fed females contained apparently mature oocytes, but their development into viable eggs was not demonstrated. Later, Boorman & Goddard (1970) demonstrated that Culicoides impunctatus can be autogenous. In C. hollensis, C. melleus and C. mississippiensis, among which field studies detected rates of fructose-positivity of > 50% in females (Magnarelli, 1981; Stewart & Kline, 1999), autogeny has been confirmed [summarized in Linley (1983)]. No evidence for autogeny is reported for C. variipennis, which according to Mullens (1985) shows low levels of sugar-feeding behaviour. The topic of autogeny in Culicoides spp. needs to be examined further, perhaps using immatures collected in the field and reared to adulthood or with newly established laboratory colonies, However, a major obstacle to these approaches is the lack of knowledge about the mating conditions of biting midges (Jamnback, 1961; Kettle, 1977; Veronesi et al., 2009). In the current study, we were able to perform laboratory experiments under realistic environmental conditions, which included a light regime and fluctuating temperatures that mimicked summer days. Usually, constant temperatures are run in experiments to determine life history parameters or vector competence (e.g. Campbell & Kettle, 1975; Wittmann et al., 2002; Lysyk & Danyk, 2007; Carpenter et al., 2011). Although the present authors are not aware of any studies comparing the effects of fluctuating and constant temperatures on life history traits, recent work has demonstrated that vector competence under a fluctuating temperature regimen can differ from that observed in conditions of constant mean temperatures (Paaijmans et al., 2010; Lambrechts et al., 2011).
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Conclusions Culicoides of the Obsoletus and Pulicaris groups are strongly dependent on natural sugar sources to increase their fitness in terms of longevity and reproduction, and these sources may thereby increase their vectorial capacity. Biting midges may represent a good target for toxic sugar-baited traps such as those already used successfully against mosquitoes (e.g. Beier et al., 2012). Such traps could easily be used in and around farm sites for local and transient biting midge control. In addition, sugar-based arbovirus surveillance methods using sugar bait stations [e.g. FTA (nucleic acid preservation) cards], as used in mosquito control (Hall-Mendelin et al., 2010; Lothrop et al., 2012), have shown promising results against Culicoides spp. (Veronesi et al., 2013) and might be further developed for the surveillance of Culicoides-borne diseases.
Acknowledgements We acknowledge the financial support of the Swiss Federal Food Safety and Veterinary Office (grant 1.08.10), the National Centre for Vector Entomology, the University of Zürich (Forschungskredit of C.K., 4974) and the Swiss National Science Foundation (SNF Professorship PP00P3_123376 to C.V.). Special thanks are extended to Jeannine Hauri, Andrea Gubler, Alexandra Blaser and Stefanie Wagner, the National Centre for Vector Entomology, the University of Zürich, Zürich, Switzerland, for their support in the field and laboratory. We also thank Holly Tuten, the National Centre for Vector Entomology, the University of Zürich, Zürich, Switzerland, for her helpful suggestions after her critical reading of the manuscript.
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Lambrechts, L., Paaijmans, K.P., Fansiri, T. et al. (2011) Impact of daily temperature fluctuations on dengue virus transmission by Aedes aegypti. Proceedings of the National Academy of Sciences of the United States of America, 108, 7460–7465. Linley, J.R. (1983) Autogeny in the Ceratopogonidae: literature and notes. The Florida Entomologist, 66, 228–234. Lothrop, H.D., Wheeler, S.S., Fang, Y. & Reisen, W.K. (2012) Use of scented sugar bait stations to track mosquito-borne arbovirus transmission in California. Journal of Medical Entomology, 49, 1466–1472. Lysyk, T.J. & Danyk, T. (2007) Effect of temperature on life history parameters of adult Culicoides sonorensis (Diptera: Ceratopogonidae) in relation to geographic origin and vectorial capacity for bluetongue virus. Journal of Medical Entomology, 44, 741–751. Magnarelli, L.A. (1981) Parity, follicular development, and sugar feeding in Culicoides melleus and C. hollensis. Environmental Entomology, 10, 807–811. McCrae, A.W.R. (1989) Differences in sugar-feeding activity between tropical and temperate mosquitoes: field observations and their implications. Vector Ecology Newsletter, 20, 16. Mitchell, C.J. & Briegel, H. (1989) Inability of diapausing Culex pipiens (Diptera: Culicidae) to use blood for producing lipid reserves for overwinter survival. Journal of Medical Entomology, 26, 318–326. Molyneux, D.H., Moore, J. & Maroli, M. (1991) Sugars in sandflies. Parassitologia, 33 (Suppl.), 431–436. Mullens, B.A. (1985) Age-related adult activity and sugar feeding by Culicoides variipennis (Diptera: Ceratopogonidae) in Southern California, U.S.A. Journal of Medical Entomology, 22, 32–37. Muller, G.C., Hogsette, J.A., Beier, J.C. et al. (2012) Attraction of Stomoxys sp. to various fruits and flowers in Mali. Medical and Veterinary Entomology, 26, 178–187. Paaijmans, K.P., Blanford, S., Bell, A.S., Blanford, J.I., Read, A.F. & Thomas, M.B. (2010) Influence of climate on malaria transmission depends on daily temperature variation. Proceedings of the National Academy of Sciences of the United States of America, 107, 15135–15139. R Development Core Team (2011) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna. Schlein, Y. & Jacobson, R.L. (1994) Mortality of Leishmania major in Phlebotomus papatasi caused by plant feeding of the sand flies. American Journal of Tropical Medicine and Hygiene, 50, 20–27. Schlein, Y. & Muller, G. (1995) Assessment of plant tissue feeding by sand flies (Diptera: Psychodidae) and mosquitoes (Diptera: Culicidae). Journal of Medical Entomology, 32, 882–887. Spencer, C.Y., Pendergast, T.H. IV & Harrington, L.C. (2005) Fructose variation in the dengue vector, Aedes aegypti, during high and low transmission seasons in the Mae Sot region of Thailand. Journal of the American Mosquito Control Association, 21, 177–181. Stanfield, T.K. & Hunter, F.F. (2010) Honeydew and nectar sugars differentially affect flight performance in female black flies. Canadian Journal of Zoology, 88, 69–72. Stewart, R.G. & Kline, D.L. (1999) Sugar feeding by Culicoides mississippiensis (Diptera: Ceratopogonidae) on the yaupon holly, Ilex vomitoria. Journal of Medical Entomology, 36, 268–271. Stone, C.M., Hamilton, I.M. & Foster, W.A. (2011) A survival and reproduction trade-off is resolved in accordance with resource availability by virgin female mosquitoes. Animal Behaviour, 81, 765–774. Van Handel, E. (1965) The obese mosquito. Journal of Physiology, 181, 478–486.
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Sugar feeding of Culicoides biting midges Van Handel, E. (1972) The detection of nectar in mosquitoes. Mosquito News, 32, 458. Venter, G.J. & Meiswinkel, R. (1994) The virtual absence of Culicoides imicola (Diptera: Ceratopogonidae) in a light-trap survey of the colder, high-lying area of the eastern Orange Free State, South Africa, and implications for the transmission of arboviruses. Onderstepoort Journal of Veterinary Research, 61, 327–340. Veronesi, E., Venter, G.J., Labuschagne, K., Mellor, P.S. & Carpenter, S. (2009) Life-history parameters of Culicoides (Avaritia) imicola Kieffer in the laboratory at different rearing temperatures. Veterinary Parasitology, 163, 370–373. Veronesi, E., Henstock, M., Gubbins, S. et al. (2013) Implicating Culicoides biting midges as vectors of Schmallenberg virus using semi-quantitative RT-PCR. PLoS ONE, 8, e57747.
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Wittmann, E.J., Mellor, P.S. & Baylis, M. (2002) Effect of temperature on the transmission of orbiviruses by the biting midge, Culicoides sonorensis. Medical and Veterinary Entomology, 16, 147–156. Xue, R.D., Barnard, D.R. & Müller, G.C. (2010) Effects of body size and nutritional regimen on survival in adult Aedes albopictus (Diptera: Culicidae). Journal of Medical Entomology, 47, 778–782. Zimmer, J.Y., Haubruge, E., Francis, F. et al. (2008) Breeding sites of bluetongue vectors in northern Europe. Veterinary Record, 162, 131. Accepted 3 July 2014
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12086
Sugar-feeding behaviour and longevity of European Culicoides biting midges C. K A U F M A N N 1 , A. M A T H I S 1 and C. V O R B U R G E R 2 1
National Centre for Vector Entomology, Institute of Parasitology, Vetsuisse Faculty, University of Zürich, Zürich, Switzerland and Institute of Integrative Biology, ETH Zürich and EAWAG (Swiss Federal Institute of Aquatic Science and Technology), Dübendorf, Switzerland 2
Abstract. Most haematophagous insect vectors can also use sugar as an energy source; thus their sugar-feeding behaviour influences their longevity and blood-feeding rate and hence their vectorial capacity. Scant information is available on the sugar-feeding behaviour of Culicoides Latreille biting midges (Diptera: Ceratopogonidae), which are vectors of bluetongue and Schmallenberg viruses. The longevity of laboratory-reared Culicoides nubeculosus (Meigen) under fluctuating temperatures (16 and 28 ∘ C) and with access to water or water and blood was on average 6.4 days and 8.9 days, respectively, which was around one third of the lifespan of siblings with access to sugar or sugar and blood (22.2 days and 27.1 days, respectively). Access to honeydew significantly increased the midge’s longevity, whereas the provision of extrafloral nectaries had no impact. Females with access to sugar produced a significantly higher number of eggs (65.5 ± 5.2) than their starved sisters (45.4 ± 8.4). More than 80% of field-caught female Culicoides from the two most abundant European groups, Obsoletus (n = 2243) and Pulicaris (n = 805), were fructose-positive. Fructose-positivity was high in all physiological stages and no seasonal variability was noted. The high rate of natural sugar feeding of Culicoides offers opportunities for the development of novel control strategies using toxic sugar baits and for the monitoring of vector-borne diseases using sugar-treated FTA (nucleic acid preservation) cards in the field. Key words. Aphids, blood, control strategies, fructose, honeydew, nectar, vectors.
Introduction Most biting flies rely on sugar sources in order to supplement their blood diet with carbohydrates. Naturally occurring sugar sources exploited by biting flies include floral and extrafloral nectaries, and fruits (damaged, rotten or dried fruits), as well as honeydew (Downes, 1958; Foster, 1995; Engel et al., 2001; Stanfield & Hunter, 2010; Muller et al., 2012). In addition, mosquitoes (Diptera: Culicidae) and sandflies (Diptera: Psychodidae) have been shown to access nutritious plant sap directly from vegetative tissues (Schlein & Jacobson, 1994; Schlein & Muller, 1995; Junnila et al., 2010). Sugar is used as a nutritional source that is additional to teneral energy reserves and can be stored as glycogen or lipid (Van Handel, 1965). This ‘sweet
source’ improves the insect’s flight performance (e.g. Kaufmann & Briegel, 2004), as well as mating rates and overall reproductive success (Gary et al., 2009; Gu et al., 2011; Stone et al., 2011). Sugar feeding also contributes to the survival of diapausing adults (Mitchell & Briegel, 1989) and to longevity in general (Jamnback, 1961; Gary & Foster, 2004; Impoinvil et al., 2004). The sugar-feeding behaviour of biting flies has been studied thoroughly, particularly in mosquitoes (reviewed in Foster, 1995), and has revealed distinct characteristics. The Asian tiger mosquito Aedes albopictus (Stegomyia albopicta) Skuse (Diptera: Culicidae) relies on sugar intake as its mean longevity without sugar stops is only a few days (Xue et al., 2010). By contrast, sugar feeding was observed to be rare in the yellow fever mosquito, Aedes aegypti (L.) (Stegomyia aegypti) (Edman
Correspondence: Alexander Mathis, National Centre for Vector Entomology, Institute of Parasitology, Vetsuisse Faculty, University of Zürich, Winterthurerstrasse 266a, CH-8057 Zürich, Switzerland. Tel.: + 41 44 635 85 36; Fax: + 41 44 635 89 07; E-mail: [email protected] © 2014 The Royal Entomological Society
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2 C. Kaufmann et al. et al., 1992), and similar observations were reported for the African malaria mosquitoes Anopheles gambiae Giles s.l. and Anopheles funestus Giles (Diptera: Culicidae) (Beier, 1996). However, sugar intake in these mosquitoes varies depending on season and natural habitat, and can be considerable (Bidlingmayer & Hem, 1973; Spencer et al., 2005; Gu et al., 2011). The European malaria vector Anopheles atroparvus Thiel is dependent on sugar sources to achieve strong flight performance, whereas the main African malaria vector, An. gambiae, reaches comparable flight distances when fed either only with sugar or only with blood (Kaufmann & Briegel, 2004). Sugar uptake can boost the vectorial capacity of mosquitoes by enhancing their longevity beyond the extrinsic incubation period of the disease agents they transmit (Xue et al., 2010). However, the biting activity of the main African malaria vector An. gambiae can increase if sugar is lacking (Gary & Foster, 2001), resulting in the earlier transmission of vector-borne disease. Clearly, knowledge about the sugar-feeding behaviour of vector species is an important component in assessments of their vectorial capacity. A few studies of the natural sugar-feeding behaviour of Culicoides biting midges, which are vectors of the bluetongue and Schmallenberg viruses, have been carried out in the U.S.A. [i.e. in Culicoides melleus (Coquillet) and Culicoides hollensis (Melander and Brues) (Magnarelli, 1981), Culicoides variipennis (Coquillet) (Mullens, 1985), and Culicoides mississippiensis Hoffman (Stewart & Kline, 1999)]. Further, wild-caught Culicoides obsoletus (Meigen) were shown to live longer in the laboratory when supplied with sugar (Jamnback, 1961). The data available on the main incriminated European vectors (i.e. species of the Obsoletus and Pulicaris groups) are scant with regard to their general biology, including their sugar-feeding behaviour (Carpenter et al., 2009). Species of the Obsoletus group are known to have breeding habitats in the close surroundings of livestock farms (Kettle & Lawson, 1952; Zimmer et al., 2008). Hence, suitable blood hosts can be found in close proximity to eclosed midges, which raises the question of whether midges rely on natural sugar sources. In the present study, the sugar-feeding behaviour of Culicoides spp. from the Obsoletus and Pulicaris groups, caught on a farm in Switzerland, was assessed. In addition, possible sugar ingestion from different sources (floral and extrafloral nectaries, honeydew and sugar solution) and its effects on longevity and reproduction were investigated using Culicoides nubeculosus (Meigen) as a laboratory model organism.
Materials and methods Insects and climatic conditions The experimental work was carried out with laboratory-reared C. nubeculosus ceratopogonid biting midges, originally kindly provided by Dr S. Carpenter, Pirbright Institute, Woking, U.K. Insects were reared as described in Boorman (1974), except that larvae were fed with pulverized Tetramin® only and blood feeding was achieved through a Nescofilm® membrane with heparinized sheep blood. In the main colony, insects were kept at a mean ± standard deviation (SD) temperature of 24 ± 0.5 ∘ C and
relative humidity (RH) of 85 ± 5% under long day conditions (LD 16 : 8 h) that included dawn and dusk periods of 1 h each. All experiments were performed in a climate chamber (Kälte 3000 AG, Landquart, Switzerland) under summer day conditions (i.e. temperature range: 16–28 ∘ C; mean ± SD temperature: 22 ± 0.5 ∘ C; RH: 60–90%; mean ± SD RH: 78 ± 5%) (Fig. 1) and a long-day setting as described above. The minimum RH of 60% was set to occur at the early afternoon peak in temperature. Indigenous biting midges were caught alive on a livestock (i.e. cows, horses and chickens) farm in Switzerland (47∘ 22′ 30.86′′ N, 08∘ 34′ 57.86′′ E). A modified Onderstepoort ultraviolet (UV) light suction trap was used for trapping, as described previously (Venter & Meiswinkel, 1994), and a cubic cage (24.5 × 24.5 × 24.5 cm3 ; BugDorm-42222F; MegaView Science Co., Ltd, Taichung, Taiwan) was used as a trapping vessel. Trapping was carried out overnight (including dawn and dusk) in spring (April and May), summer (June and August) and autumn (late September). After trapping, cages were placed in thermally insulated containers chilled with ice and transported to the nearby institute where the insects were killed at −20 ∘ C and then sorted into Obsoletus and Pulicaris group specimens. Although no attempts were made to identify the investigated Culicoides to species level, the most likely species at this site, as deduced from the identification by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry of midges collected from comparable sites (Kaufmann et al., 2012) were C. obsoletus, Culicoides scoticus Downes and Kettle (Obsoletus group) and Culicoides pulicaris (L.), Culicoides lupicaris Downes and Kettle, and Culicoides grisescens Edwards (Pulicaris group). Females were additionally divided into the physiological stages of nulliparous, parous, gravid and blood-fed. If they were not to be immediately processed for fructose analysis, insect samples were stored at −80 ∘ C.
Longevity with experimental diets Longevity experiments were performed in cubic cages (17.5 × 17.5 × 17.5 cm3 ; BugDorm-41515) under five different diets: (a) no nutrition; (b) water; (c) 10% sucrose solution; (d) water and blood, and (e) 10% sucrose solution and blood. Water and sucrose solutions were available ad libitum; heparinized sheep blood was provided daily through a Nescofilm® membrane for 30 min. The experiments were repeated in triplicate. To start the experiment, 10 newly eclosed C. nubeculosus of each sex were transferred to each experimental cage. Survival was monitored daily; dead individuals were removed and their sex determined. Every second day, eggs laid on a wet filter paper in the cage were removed and discarded.
Longevity and sugar ingestion with natural sugar sources Two experiments were carried out to investigate the midges’ capability to use naturally occurring sugar sources and their effect on longevity. The first experiment comprised four treatments. All cages (BugDorm-42222F) were supplied with a
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12086
Sugar feeding of Culicoides biting midges
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Fig. 1. Temporal temperature ( ) and humidity ( ) profiles for simulated summer days.
non-flowering broad bean plant (Vicia faba), a vetch producing extrafloral nectaries on leaf stipules (Engel et al., 2001), on which the presence of extrafloral nectaries was manipulated (left intact or stipules removed with forceps), with or without the presence of honeydew-producing black bean aphids (Aphis fabae Scopoli) (Hemiptera: Aphidae) in a 2 × 2 factorial design. The presence and absence of aphids (providing honeydew) and/or of nectar in extrafloral nectaries were visually confirmed. Each treatment was replicated three times. A first set of 10 male and 10 female C. nubeculosus was added to the cages for 1 day, then removed and analysed for fructose ingestion using the cold anthrone test (see below). A second set of midges was then added to these cages (with permanent access to the different natural sugar sources) to monitor longevity as described under ‘Longevity with experimental diets’ above. In the second experiment, midges were given access to two spring flowering plants (the daisy, Bellis perennis and the lesser celandine, Ranunculus ficaria) for 1 day. Control cages contained neither food nor water. The daisy treatment was replicated in four cages, the lesser celandine treatment in three cages and the control treatment in two cages.
Fecundity Female and male C. nubeculosus were maintained in cubic cages (BugDorm-41515) with permanent access to water only or to 10% sucrose. One single bloodmeal (heparinized sheep blood) was given when females were 3 days old; only females that were visually determined using a stereomicroscope to be fully engorged were included in the further trial. After an additional 3 days of incubation time, females were dissected in order to count their eggs (n = 24 females for each treatment).
pellet pestles (Fisher Scientific AG, Wohlen, Switzerland). Each cohort of samples tested included two negative controls and a standard of 0.1% fructose (Sigma-Aldrich Chemie GmbH) in ethanol (25%). A preliminary experiment was performed using the in-house C. nubeculosus colony. Twenty-five insects with access to water only were all found to be negative in the cold anthrone test, whereas 96% of insects with access to 10% fructose solution for 1 h (after being deprived of water for 6 h) were positive (visual observation of colour changes from yellowish to green).
Statistical analysis Survival data were analysed with Cox proportional hazards models using the open source statistical software R Version 2.13.0 (R Development Core Team, 2011), testing for the effects of sex and the different treatments on survival. To account for the non-independence of midges developing in the same cages, we used the coxme library, a contributory package to R for Cox mixed-effects models, which allowed us to enter ‘cage’ as a random effect in our models. The data on sugar ingestion in the experiments were treated as binomial (fructose-negative or fructose-positive) and analysed using generalized linear mixed models (GLMMs) with the logit link and binomial errors, executed with the lme4 library in R. Again, the effects of sex were tested for in the different treatments and ‘cage’ was included as a random effect to account for the non-independence of biting midges kept in the same cage. Tukey contrasts were used for multiple comparisons of means as implemented in the multcomp library (Hothorn et al., 2008).
Results Fructose-sensitive cold anthrone test
Longevity with experimental diets
The presence of fructose was determined using the cold anthrone test according to Van Handel (1972). In brief, whole insects were homogenized in 0.2 mL of anthrone reagent (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) using 1.5-mL microtubes with a hand-homogenizer and disposable
Culicoides nubeculosus were kept under five different diets (no food, water, sucrose solution, and blood feeding with access to water or sucrose), and all experiments were repeated three times. Overall, mortality was higher in males than in females in this experiment (Z = 2.36, P = 0.018). Although there was
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12086
4 C. Kaufmann et al. Table 1. Longevity of Culicoides nubeculosus biting midges∗ under climatic summer day conditions† and different experimental diets (water, sucrose, blood). Longevity, days
Longevity with natural sugar sources
Diet
Sex
Mean
Median
Maximum
NF
♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂
4.27 3.70 6.40 5.27 8.87 5.63 22.17 17.43 27.07 16.20
4.00 4.00 6.50 5.00 8.00 5.00 18.00 15.00 27.50 15.00
5 5 8 6 13 8 51 30 40 28
WF WBF SF SBF
to a positive effect on female survival (Table 1, Fig. 2) because males do not feed on blood.
∗n = 10 males and 10 females per treatment, repeated in triplicates. †16–28 ∘ C, 60–90% relative humidity, light regimen LD 16 : 8 h. NF, no food; WF, water-fed; WBF, water and blood-fed; SF, sucrose-fed; SBF, sucrose and blood-fed. Water and 10% sucrose solution were available ad libitum; blood-feeding through an artificial membrane was offered daily for 30 min.
significant variation in mortality rates among cages within treatments (accounted for by the random cage effect in the model: 𝜒 2 1 = 13.88, P < 0.001), clear differences among food treatments on mortality were evident. Midges with access to sucrose in their diet lived considerably longer than those with no access to sucrose (Table 1, Fig. 2), independent of the additional availability of blood (sucrose-fed vs. sucrose and blood-fed treatment: Z = −0.104, P = 0.999). Mortality was highest in the no-food (NF) treatment (Table 1, Fig. 2). Access to water only (WF) decreased mortality significantly (NF vs. WF: Z = −6.22, P < 0.001), and access to blood in addition to water (WBF treatment) decreased mortality further (WF vs. WBF: Z = 3.38, P = 0.006). Not surprisingly, this effect was mainly attributable
In the first experiment with natural sugar sources (extrafloral nectaries; honeydew produced by black bean aphids), C. nubeculosus males died earlier than females (Z = 3.32, P < 0.001) but there was no significant variation in mortality rates among cages within treatments (𝜒 2 1 = 0.46, P = 0.498). Inspection of longevities and survivorship curves (Table 2, Fig. 3) clearly shows that the presence of aphids and thus access to their excreted honeydew strongly reduced mortality (Z = −6.23, P < 0.001), whereas the presence of extrafloral nectaries did not significantly improve survival (Z = −1.51, P = 0.130). There was no significant interaction between the presence of aphids and extrafloral nectaries on midge mortality (Z = 1.47, P = 0.140).
Sugar-feeding behaviour in the laboratory and field Fructose analysis with the cold anthrone test confirmed that C. nubeculosus ingested aphid honeydew but very little nectar from plant extrafloral sources. Thus, in the presence of aphids, 68–89% of midges ingested sugar (Fig. 4), but only 3–17% were fructose-positive in the absence of aphids. Accordingly, aphid presence was the only significant effect in the model (Z = 3.24, P = 0.001) and the presence of extrafloral nectaries had no significant effect (Z = 0.63, P = 0.532). Males overall tested positive for fructose more frequently than females, but this difference was not statistically significant (Z = 1.59, P = 0.113). Two common blooming flowers (the daisy and lesser celandine) were also tested as potential natural sugar sources for C. nubeculosus and were found to differ markedly in their suitability. Only a few (7% of males) of the laboratory biting
Fig. 2. Longevity of male and female Culicoides nubeculosus biting midges kept under summer day (16–28 ∘ C, 60–90% relative humidity, light regimen LD 16 : 8 h) climatic conditions on different diets. , no food; , water-fed; , water and blood-fed; , sucrose-fed; , sucrose and blood-fed. © 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12086
Sugar feeding of Culicoides biting midges
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Table 2. Longevity of Culicoides nubeculosus biting midges∗ under climatic summer day conditions†and different sources (natural sugars). Longevity, days Source
Sex
Mean
Median
Maximum
N− /A−
♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂
6.67 5.62 7.11 5.90 13.36 8.37 12.60 9.73
7.00 5.00 7.00 6.00 14.50 8.00 12.00 9.50
8 7 10 9 20 16 18 14
N+ /A− N− /A+ N+ /A+
∗n = 10 males and 10 females per treatment, repeated in triplicates. †16–28 ∘ C, 60–90% relative humidity, light regimen LD 16 : 8 h. N− /N+ : without or with access to extrafloral nectaries on field bean plants Vicia faba; A− /A+ : without or with infestation with the black bean aphid Aphis fabae producing honeydew.
midges accessed the nectar of daisies, whereas 58% of tested females and 67% of tested males accessed the lesser celandine nectar (Fig. 4). All control midges with access to water only were fructose-negative (Fig. 4). The GLMM to compare sugar ingestion between midges with access to daisies and lesser celandine flowers, respectively, did not converge because no females from daisy cages tested positive. To make the model converge and obtain a conservative test for the difference between flowers, we artificially set one female from a daisy cage as fructose-positive. This conservative comparison still showed a highly significant difference (Z = 3.61, P < 0.001), whereas the difference in sugar ingestion between males and females was not statistically significant (Z = 0.78, P = 0.437). At a farm, we collected 2243 Culicoides of the Obsoletus group (2169 females, 74 males) and a total of 805 specimens of the Pulicaris group (796 females, 9 males), all of which
Fig. 4. Percentages of fructose-positive male (black bars) and female (white bars) Culicoides nubeculosus biting midges after 24 h of access to the broad bean plant Vicia faba without (N− ) or with (N+ ) extrafloral nectaries and without (A− ) the black bean aphid Aphis fabae (N− /A− ; N+ /A− ) or with access to V. faba (without or with extrafloral nectaries) infested with (A+ ) A. fabae (N− /A+ ; N+ /A+ ). In addition, both sexes of C. nubeculosus were tested with access to daisies (D), Bellis perennis, lesser celandines (P), Ranunculus ficaria, or under non-feeding conditions (n = 10 males/females, repeated in triplicates; climatic summer day conditions: 16–28 ∘ C, 60–90% relative humidity, LD 16 : 8 h).
were individually subjected to the anthrone test. In both groups, the percentage of fructose-positive individuals was higher in females than in males [Obsoletus: 83% vs. 66% (𝜒 2 1 = 12.77, P < 0.001); Pulicaris: 80% vs. 33% (𝜒 2 1 = 9.213, P = 0.002)] (Table 3), but the figures for males were obtained from much lower numbers of individuals. Sugar-feeding behaviour also differed among the physiological stages of females in both groups (Obsoletus: 𝜒 2 3 = 51.50, P < 0.001; Pulicaris: 𝜒 2 3 = 9.91,
Fig. 3. Longevity of male and female Culicoides nubeculosus biting midges kept at summer day (16–28 ∘ C, 60–90% relative humidity, light regimen LD 16 : 8 h) climatic conditions with permanent access to the field bean plant Vicia faba with (N+ ) or without (N− ) extrafloral nectaries and with (A+ ) or without (A− ) infestation with the black bean aphid Aphis fabae. , N− /A− ; , N+ /A− ; , N− /A+ ; , N+ /A+ . © 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12086
6 C. Kaufmann et al. Table 3. Percentages of fructose-positive Culicoides spp. caught at a farm site in Switzerland. Anthrone-positive samples, % (total n investigated) Stage Group
Sex
Nulliparous Parous Gravid Blood-fed ♀
Obsoletus 86 (1348) Pulicaris 83 (506)
83 (442) 77 (270)
70 (371) 59 (17)
88 (8) 100 (3)
♂
83 (2169) 80 (796)
66 (74) 33 (9)
Table 4. Seasonal prevalences of fructose-positive female Culicoides spp. caught at a farm site in Switzerland. Anthrone-positive samples, % (total n investigated) Group
Spring
Summer
Autumn
Obsoletus
88 (4410) 83 (120)
82 (1539) 81 (614)
82 (189) 73 (62)
Pulicaris
P = 0.019) (Table 3). The percentage of fructose-positive insects was around 80% or more in all stages, with the exception of gravid females (70 and 59% for the Obsoletus and Pulicaris groups, respectively). However, the numbers of gravid (n = 17 in the Pulicaris group) and blood-fed (n = 8 in the Obsoletus group, n = 3 in the Pulicaris group) females were very low. There was little evidence for any seasonality in the sugar-feeding behaviour of Culicoides at this farm site. Although the percentages of fructose-positive females differed significantly among the spring, summer and autumn samples (Obsoletus: 𝜒 2 2 = 8.99, P = 0.011; Pulicaris: 𝜒 2 2 = 16.18, P > 0.001), the values were consistently high throughout the year (Table 4).
Egg production with access to sucrose or under starvation To assess a potential effect of sugar availability on fecundity, a single bloodmeal was provided to midges. Dissection of 24 females per group showed that females with access to 10% fructose contained a significantly higher amount of eggs (65.5 ± 5.2) than their starved siblings (45.4 ± 8.4), which had access to water only (P < 0.001, two-sided t-test).
Discussion The high percentage (around 80% or more) of fructose-positive Culicoides of both Obsoletus and Pulicaris group species, collected at a farm site, was not expected. As the anthrone test used is known to more reliably detect recent sugar feeding (Mullens, 1985), these findings strongly indicate that these Culicoides, which are most abundant around farm sites in Central Europe,
frequently access natural sugar sources. In addition, the high level of sugar feeding among all the different life stages of the field-caught midges (nulliparous, parous, gravid and blood-fed) (Tables 3 and 4) confirms the necessity to access natural sugar sources throughout the lifespan. Seasonal and environmental factors have been shown to influence sugar intake behaviour in mosquitoes (Bidlingmayer & Hem, 1973; Spencer et al., 2005; Gu et al., 2011). However, in the present study, no differences were observed between the seasons evaluated (late spring, summer, autumn). During the cold winter months in Switzerland, there is barely any Culicoides activity (Kaufmann et al., 2009). Levels of fructose-positivity of 66 and 64% were identified in the saltwater marsh species C. melleus and C. hollensis, respectively, when caught with an aspirator upon landing or when blood feeding on humans (note that host blood does not contain fructose) (Magnarelli, 1981). Fructose-positivity of 56% was found in C. mississippiensis collected by sweeping the midges directly from their principal sugar source, the flowering yaupon holly (Ilex vomitoria) (Stewart & Kline, 1999). By contrast, in a study conducted in southern California, only 12% of nulliparous and 24% of parous females of C. variipennis were found to be fructose-positive throughout the year, with the highest percentages occurring between December and May (Mullens, 1985). This suggests rather low sugar consumption, but may be explained by the fact that the midges were caught with carbon dioxide-operated Centers for Disease Control (CDC) miniature traps without light, which mainly attract host-seeking midges. By contrast, the low level of sugar-feeding behaviour in C. variipennis may reflect facultative sugar consumption. A similar strategy was shown in An. gambiae, which survive and fly well on blood alone when natural sugar sources are rare as long as plenty of human hosts are available (McCrae, 1989; Kaufmann & Briegel, 2004), but benefit from additional sugar sources (Gary & Foster, 2001, 2004). The lifespan of Culicoides was substantially increased with access to sugar, as shown for C. nubeculosus in this laboratory study, confirming earlier findings with field-collected C. obsoletus (Jamnback, 1961) and Culicoides brevitarsis (Campbell & Kettle, 1975). Culicoides nubeculosus with access to blood, but not sugar, exhibited short lifespans of about 1 week in our experiments, similar to the observations described for C. obsoletus (Jamnback, 1961). With regard to the transmission of pathogens, a lifespan of 1 week would be sufficient at higher temperatures. The extrinsic incubation period for bluetongue virus in Culicoides sonorensis was shown experimentally to be < 5 days at temperatures of > 20 ∘ C (Carpenter et al., 2011). Thus, virus transmission by Culicoides spp. seems possible without an additional sugar supply. However, in vector competence studies, biting midges have been maintained with permanent access to sugar solution (e.g. Carpenter et al., 2011) and do not need to fly long distances in the laboratory (e.g. to find hosts or breeding sites), which would require additional energy in the field. Our laboratory and field results demonstrated that blood alone is far from optimal for survival and the accumulation of maternal reserves. Hence, we conclude that sugar stops increase the vectorial capacity of biting midges by extending their longevity and the higher potential number of bloodmeals taken during their lifespan.
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12086
Sugar feeding of Culicoides biting midges Interestingly, males of C. nubeculosus showed a slightly higher prevalence of sugar ingestion than females under all experimental conditions (Fig. 4). By contrast, in all field studies, males were consistently less frequently fructose-positive than females (Mullens, 1985; Stewart & Kline, 1999), a finding that held true in this study in field-caught males. Fructose-positivity in males decreased during dusk, which may be related to their mating and flight activity (Stewart & Kline, 1999). Thus, in laboratory cages, reduced flight activity in males may result in lower energy consumption and therefore in a higher probability that fructose-positive insects will be detected. The current study showed that floral nectar and honeydew, which are important sugar sources for mosquitoes (Foster, 1995), sandflies (Molyneux et al., 1991) and blackflies (Stanfield & Hunter, 2010), can be accessed and utilized easily by C. nubeculosus, and result in increased longevity, whereas other sources such as extrafloral nectaries or plant sap were rarely or not accessed. However, we cannot exclude the possibility that such sources are occasionally used by Culicoides, and more potentially relevant plant species should be tested because mosquitoes and sandflies have previously been found to show different preferences in accessing plant tissues from 15 different plant species (Schlein & Muller, 1995). The frequent sugar consumption of field-caught Culicoides spp. may possibly indicate autogenous reproduction for the first batch of eggs, which is known in only a few Culicoides species (summarized in Linley, 1983). However, few data about autogeny are available for species of the Obsoletus and Pulicaris groups. Jamnback (1961) suggested autogeny for C. obsoletus because unmated sugar-fed females contained apparently mature oocytes, but their development into viable eggs was not demonstrated. Later, Boorman & Goddard (1970) demonstrated that Culicoides impunctatus can be autogenous. In C. hollensis, C. melleus and C. mississippiensis, among which field studies detected rates of fructose-positivity of > 50% in females (Magnarelli, 1981; Stewart & Kline, 1999), autogeny has been confirmed [summarized in Linley (1983)]. No evidence for autogeny is reported for C. variipennis, which according to Mullens (1985) shows low levels of sugar-feeding behaviour. The topic of autogeny in Culicoides spp. needs to be examined further, perhaps using immatures collected in the field and reared to adulthood or with newly established laboratory colonies, However, a major obstacle to these approaches is the lack of knowledge about the mating conditions of biting midges (Jamnback, 1961; Kettle, 1977; Veronesi et al., 2009). In the current study, we were able to perform laboratory experiments under realistic environmental conditions, which included a light regime and fluctuating temperatures that mimicked summer days. Usually, constant temperatures are run in experiments to determine life history parameters or vector competence (e.g. Campbell & Kettle, 1975; Wittmann et al., 2002; Lysyk & Danyk, 2007; Carpenter et al., 2011). Although the present authors are not aware of any studies comparing the effects of fluctuating and constant temperatures on life history traits, recent work has demonstrated that vector competence under a fluctuating temperature regimen can differ from that observed in conditions of constant mean temperatures (Paaijmans et al., 2010; Lambrechts et al., 2011).
7
Conclusions Culicoides of the Obsoletus and Pulicaris groups are strongly dependent on natural sugar sources to increase their fitness in terms of longevity and reproduction, and these sources may thereby increase their vectorial capacity. Biting midges may represent a good target for toxic sugar-baited traps such as those already used successfully against mosquitoes (e.g. Beier et al., 2012). Such traps could easily be used in and around farm sites for local and transient biting midge control. In addition, sugar-based arbovirus surveillance methods using sugar bait stations [e.g. FTA (nucleic acid preservation) cards], as used in mosquito control (Hall-Mendelin et al., 2010; Lothrop et al., 2012), have shown promising results against Culicoides spp. (Veronesi et al., 2013) and might be further developed for the surveillance of Culicoides-borne diseases.
Acknowledgements We acknowledge the financial support of the Swiss Federal Food Safety and Veterinary Office (grant 1.08.10), the National Centre for Vector Entomology, the University of Zürich (Forschungskredit of C.K., 4974) and the Swiss National Science Foundation (SNF Professorship PP00P3_123376 to C.V.). Special thanks are extended to Jeannine Hauri, Andrea Gubler, Alexandra Blaser and Stefanie Wagner, the National Centre for Vector Entomology, the University of Zürich, Zürich, Switzerland, for their support in the field and laboratory. We also thank Holly Tuten, the National Centre for Vector Entomology, the University of Zürich, Zürich, Switzerland, for her helpful suggestions after her critical reading of the manuscript.
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