SEPTEMBER1989 Monrelrrv IN Aour,t C. venapsNttts 387

ENVIRONMENTAL FACTORS AFFECTING MORTALITY OF ADULT CULrcOIDES VARIIPENNIS (DIPTERA: )IN THE LABORATORY

GREGG J. HUNT, WALTER J. TABACHNICK eNo CHARLES N. McKINNON

U.S. Departmcnt of Agrbulture, Agrirultural ResearchSeruice, -borrw DiseasesResearch Laboratory,P.O. Bor 3965,Lararnie, WY 82071-3965

ABSTRACT. The effects of severalenvironmental factors on mortality of adult Culicoi.d,esuariipennis in the laboratory were evaluated.Daily mortality rates significantly increasedwhen adult midges were maintained at an elevated constant temperature (26'C). Adult C. uariipennis handled the least during routine maintenance procedures had the lowest daily mortality rate. Survival was not sigrrificantly affectedby varying density levelsof adult midgesin the sizeof container routinely usedin our laboratory. Inrplications of these observationsusing adult C. uariipennis for future studies are discussed.

INTRODUCTION (1978) showedthat adult size in female C. uari- ipennis, as measured by dry weight and wing (Qo- The biting midge, Culicoidesuariipennis Iength, was inversely proportional to both larval primary quillett), is the vector of bluetongue density and rearing temperature. In addition, (BTV) virus in , and wild ruminants, larger female adults lived longer and produced and of epizootic hemorrhagic diseasevirus in more eggs than did smaller adults. Mullens deer and cattle in the U.S.A. Bluetonguedisease (1987)observed a seasonaltrend of adult size in is a serious economic threat to domestic rumi- female C. uariipennis, with an apparent inverse nants. It causesmortality in sheep,reproductive relationship between wing length and air tem- impairment in both sheep and cattle, and re- perature near the 's natural habitat. The strictions in the international movement of rate of developmentof immature C. uariipennis germplasm (Gibbs breeding Iivestock and and has also been inversely related to temperature Greiner 1988,Holbrook 1988). (Akey et al. 1978, Mullens and Rutz 1983, Procedures have been developed for mass Vaughanand Turner 1987). rearing C. uariipennis to conduct entomologic In the present study environmental factors, as a vector studies, to evaluate this insect of such as temperature, blood feeding, adult den- importance severaldisease agents of veterinary sity per container, and insect-handling tech- and to develop insect control strategies at the niques,which might affect the mortality of adult Arthropod-borne Animal Diseases Research C. uariipennis in the laboratory, were evaluated. Laboratory (Jones 1960,1964, Jones et al. 1969, Akey et al. 1978,G. J. Hunt, unpublished data). procedures However, similar rearing have MATERIALS AND METHODS proved inadequate for maintaining many freld- collected populations of immature or adult C. Test : Culicoidesvariipennis were ob- uariipennis. Adults of these populations have tained from the AA colony (establishedDecem- survived poorly during extended holding pe- ber 1957 from Edwards County, Texas) (Jones riods, such as the extrinsic incubation period 1960) and the AK colony (established August (10-14 days) required for BTV transmission 1973 from Owyhee County, Idaho) (Jones and studies. Excessivemortality has been observed Foster 1978).Both colonieshave been contin- with various speciesof field-collected ually maintained without the addition of wild C. during vector competencestudies (Jones et al, uariipennis since establishment. Insect-rearing 1983,Mullen et al. 1985).Environmental factors proceduresfor massproduction were used (G. J. (i.e., insect-rearing procedures)may contribute Hunt, unpublished data). ro high mortality in these natural populations Experimental design: Male and female adult ., <,. uariipennls brought into the laboratory. C. uariipennis,24- to 48-hr old, were randomly Of environmental factors which influence im- divided into two groups from each colony. In- rrortant life history traits of field-collected and sects in the first group (A) were anesthetized la.oratory-reared C. uariipennis, only tempera- with medical-gradeCO2 gas and placedin blood- ture and insect density during larval develop- feeding cages. Teneral female adults were al- ment have been studied in detail. Wing length lowed to feed on defrbrinated sheepblood with- in female C. uariipennis has been shown to be out additives through membranes prepared from inversely proportional to rearing temperature the skins of 1- to 3-day-old chicks using an (Hensleighand Atchley 1977,Mullens and Rutz artificial blood-feeding apparatus (G. J. Hunt 1983, Vaughan and Turner 1987). Akey et al. and C. N. McKinnon, unpublished data). After 366 Jouruu0['Tr{D Aunmcm MosQurro Collrnor Assocnuoll VoL.b, N0. 3

2.5 h of feeding, insects were anesthetized and Data analysis;Mortality rates and the logro(x sorted by sex and degree of blood feeding on * 1) transformations for normality were ana- refrigerated tables with the aid of dissecting lyzed by a three-way analysis of variance (AN- microscopes. One-hundred fully-engorged ovA). midges from each colony were placed in 0.24- liter (0.5-pint) cardboard ice cream containers RESULTS with fine-mesh nylon organdycoverings. In each container, a moist substrateof absorbentcotton. Only the results of the mortality rates of adult filter paper and deionizedwater placedin a small C. uariipennis are presented in Table 1 since dish was provided for oviposition, and a vial both ANOVA analysesshowed the sameresults. with a cotton dental wick containing 10% su- Severalenvironmental factors had a significant crosesolution was alternately provided as a car- effect on daily mortality rates. bohydrate sourcefor one day followed by deion- A significant difference was detectedbetween ized water for two days. Three containers (rep- the two constant temperaturesfor either colony licates) from each colony were maintained at (Table 1). Daily mortality was higher at 26"C constanttemperatures of either 20 + l"C ot 26 than at 20"C (Fig. 1). Since no differencesin + 1"C, 40=50%RH and a 13L:11Dphotoperiod. mortality rates were detected between the two The oviposition substrates and cotton wicks colonies, these results were combined and were remoistenedeach day. On the fourth day, showedsignificant differencesamong the three the oviposition dishes with eggs were replaced handling techniques(Table 1). The highest daily with empty dishes. The second group (B) of mortality rate occurred among adult midges insects was processed and maintained in the handledthe most (group A) (Fig. 2). Anesthetiz- samemanner, exceptthese teneral female adults ing and blood feeding presumably contributed were not anesthetizednor provided blood meals to this higher mortality rate. Adult midges han- or oviposition substrates. For the third group dled the least (i.e., female adults emerging from (C) of insects, pupae frorn each colony were pupaein the containers)(group C), experienced obtained two days prior to the initial setup of the lowest daily mortality rate (Fig. 2). The the first two groups of insects. Female pupae combined mean cumulative mortality rates for were sorted with the aid of a dissecting micro- the two colonies were 52 + 4Vo and.73 + 37oat scopeand placed in 0.24-liter containerswith 20"C and 26"C, respectively.The ANOVA re- organdy coverings.Each container accommo- sults revealedsignificant differences in mortal- dated 110pupae (to compensatefor an expected ity rates from day to day (Table 1). A significant 91% adult-emergencerate) placed on a moist handling by day interaction (Table 1) was also substrate of absorbent cotton and deionized revealed;the significant differencesin mortality water in a small dish for emergence.Three con- rates due to the amount of handling were not tainers (replicates)from eachcolony were main- consistent, but changed from day to day (Fig. tained under the samerearing conditions as with 2). There were no significant differences noted the first two groups of insects. On the second between the four density levels of adult midges day, the emergencedishes were replaced with per container(F : 0.296;df : 3,173;P > 0.05). empty dishes.In summary,the varying handling techniques for the three groups of adult C. uar- DISCUSSION iipennis included anesthetizing, blood feeding, anesthetizing,chilling and sorting for group A; The mortality rate in adult C. uariipennis (or anesthetizing,chilling and sorting for group B; other vector species)during the extrinsic incu- and nothing for group C. bation period for virus transmission is an im- The effect of varying densitiesof adult insects on mortality of C. uariipennis in the 0.24-liter containers was also studied. Three containers Table1. Analysisof variancefor the effectsof environmentalfactors on mortalityrates of adult (replicates)of 100, 75, 50 and 25 blood-fedfe- males per container from the AA colony were processedin the same manner as those midges Source df F Probability -r in group A and were maintained at 20 1'C. Temperature T t4.7to 0.0001 AII containers in both experiments were ex- Colony I 0.006 0.9400ns amined daily; dead insects were counted, re- Handling z 12.440 0.0001 moved and discardedfrom each container. The Day t4 2.286 0.0080 daily mortality rate, the number of dead female Handling x day 28 2.664 0.0001 adults divided by the total number of female Interactions (other) ab adults present on that day, was determined in Error 437 eachcontainer for 15 davs. ns : not significant. MoRteLtrv IN Aoulr C. vtnttPsvNts 389

0 1 2 3 4 5 6 7 8 9 10 1'l 12 13 14 15 Day Fig. 1. Effects of two constant temperatureson daily mortality rates of adult Culicoidesuariipennis from two laboratory colonies.Points representthe averagepercent (n : 3 replicates) of daily mortality. portant factor in conducting vector competence atures on mortality rates of adult C. uariipennis studies with disease agents. Poor survival of was studied under controlled laboratory condi- infected biting midges during this incubation tions, temperature is not uniform throughout period will limit the number of C. uariipennis the environment of the insect's natural habitat. for subsequentfeeding and, therefore, affect the The effect of increasedhandling on mortality dynamics of virus transmission. rates of adult C. uariipennis is of interest to Although mortality records from field or lab- investigators using this biting midge to study oratory studies are relatively few for speciesof vector competence. The data presented here adult Culicoides, an increase in mortality at clearly show that the number of surviving in- higher temperatures was expected. Similar ob- sectswill be enhancedifhandling during routine servations have been reported for a variety of maintenance procedures is minimized. It was insect species;the cause of this relationship is expected that the effect of handling might be complex and is attributed to a variety of factors similar to the effect of density or overcrowding (Sohal 1976).One significant factor is the influ- since each increasesthe chanceof physical in- ence of temperature on the insect's metabolic jury to adult C. uariipennis.Howevet, our results activity. In general,the metabolic rate tends to did not support this hypothesisusing the density be inversely related to longevity (Sohal 1986). levels as previously described.Significantly dif- Even though the influence of constant temper- ferent daily mortality rates were not observed 390 JounNe,r,oF THEAuuRrclu Moseurro CoNrnor,Assocrarrox VoL.5,No.3

----r---. handlingA handlingB handlingC

= E ,I o t = , ir c o L- o "r---...1 o /i D. /|

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Day Fig. 2. Effects of three different handling techniqueson the combineddaily mortality rates of adult Culicoides uariipennis from two laboratory colonies. Points represent the averagepercent (n : 6 replicates) of daily mortality. A : anesthetizing,blood feeding,anesthetizing, chilling, sorting; B : anesthetizing,chilling, sorting; C : none.

among the varying density levels of adult C. Gibbs, E. P. J. and E. C. Greiner. 1988. Bluetongue uariipennis in the size of container routinely and epizootic hemorrhagicdisease, pp. 39-70.In; T. used in our laboratory. P. Monath (ed.),The :epidemiology and The vector competenceof natural populations ecology.Vol. 2. CRC Press,Inc., Boca Raton, FL. of any vector species(i.e., C. uariipennis) is Hensleigh,D. A. and W. R. Atchley. 1977.Morpho- metric variability in natural and Iaboratory popu- dependenton a variety of extrinsic and intrinsic Iations of CuLicoidesuariipennis (Diptera: factors. Cerato- The use of simple models to predict the pogonidae).J. Med. Entomol. 14:379-386. vector competenceof geographicpopulations is Holbrook, F. R. 1988.Bluetongue in the United States: fraught with difficulties. For example, although status, transmission, and control through vector high densities of a vector population are usually suppression.Bull. Soc.Vector Ecol. 13:350-353, viewed as increasing the risk of disease,there Jones, R. H. 1960. Mass-production methods for the actually may be fewer insects transmitting dis- colonization of Culicoidesuariipennis sonorensis.J. easeagents due to poor survival during the ex- Econ.Entomol. 53:731-735. trinsic incubation period at high temperatures. Jones,R. H. 1964.Mass production methodsin rearing Culicoidesuariipennis (Coquillett). Bull. W. H. O. 3I:57I-572. ACKNOWLEDGMENTS Jones,R. H., H. W. Potter and S. K. Baker. 1969.An improved larval medium for colonized Culicoides We acknowledge J. A. Kempert and D. K. uariipnnis. J. Econ. Entomol. 62:1483-1486. Virchow for technical assistancein rearing the Jones, R. H. and N. M. Foster. 1978. Relevance of laboratory insect colonies and F. R. Holbrook laboratory colonies of the vector in re- for advice and support. We thank D. H. Akey, search-Culicoidcs uariipennis and bluetongue. Am. J. Trop. Med. Hyg.27:168-177. J. R. Linley and B. A. Mullens for constructive Jones,R. H., E. T. Schmidtmannand N. M. Foster. review of the manuscript. 1983.Vector-competence studies for bluetongueand epizootic hemorrhagic disease viruses with Culi- REFERENCES CITED coides uenustus (Ceratopogonidae). Mosq. News 43:184-186. Akey,D. H., H. W. Potterand R. H. Jones.1978. Mullen, G. R., R. H. Jones,Y. Bravermanand K. E. Effectsof rearingtemperature and larval density on Nusbaum. 1985.Laboratory infections of Culiroi.des Iongevity,size, and fecundityin the biting gnat debilipalpis and C. stellifer (Diptera: Ceratopogoni Culicoidesuariipennis. Ann. Entomol. Soc. Am. dae) with bluetongue virus, pp. 239-243. In: T. L. 71:411-418. Barber and M. M. Jochim (eds.), Bluetongue and SnpreNaepn1989 MoRTALTTYrN Aour,r C. vanilpsMitrs 391

related orbiviruses. Vol. 178, Progress in Clinical Sohal, R. S. 1976.Metabolic rate and life span. Inter- and Biological Research.Alan R. Liss, Inc., New discip. Top. Gerontol. 9:25-40. York. Sohal, R. S. 1986.The rate of living theory: a contem- Mullens,B. A. 1987.Seasonal size variability inCuli' porary interpretation, pp. 23-44. 1n.'Collatz, K.-G. coides uariipennls (Diptera: Ceratopogonidae) in and R. S. Sohal (eds.),Insect aging.Springer-Verlag, southern California. J. Am. Mosq. Control Assoc. Berlin. 3:512-513. Vaughan, J. A. and E. C. Turner, Jr. 1987. Develop- Mullens,B. A. and D. A. Rutz. 1983.Development of ment of immature CuLicoi.d.esuariipennis (Diptera: immature Culicoi.desuariipennis (Diptera: Cerato- Ceratopogonidae)from Saltville, Virginia, at con- pogonidae) at constant Iaboratory temperatures. stant laboratory temperatures. J. Med. Entomol. Ann. Entomol. Soc.Am. 76:747-751. 24:390-395.