Journal of the American Control Association, 16(3):248-253,2OOO Copyright @ 2000 by the American Mosquito Control Association, Inc.

EFFECTS OF PHOTOFRIN II' ON ADULTS OF ERETMAPO D IT ES QUI N QUEVITTATU S

A. M. HELLECK lNo W. K. HARTBERG

'fX Baylor University, Department of Biology, PO Box 97388, Waco, 76798-7388

ABSTRACT, This research was conducted to further assess the efficacy of photosensitizers as adult mosquito control agents. Fourth-stage larvae of Eretmapodites quinquevittatus were fed Photofrino porfimer sodium (PII) (<3.75 pglml) and the resulting adults (parentals) were photoiffadiated. These parentals were mated with un- treated controls and resulting lst generation (F,) progeny were inbred. Comparisons of fecundity and fertility were made between parentals and resulting F, progeny. Interbred F, adults survived significantly longer than parentals (P > 0.05). Parental females laid fewer eggs than F, females, and parental eggs hatched at a signifi- cantly lower rate than eggs of F, females (P < 0.01). Neither PII nor any effects of PII seem to be transferred from parent to offspring. Reduced fecundity and fertility may be due to reduced energy levels caused by exposure to PII. Experimental adults fed PII exhibited significantly reduced survivorship when compared to untreated controls (P < 0.001). Fertility did not differ when compared to controls, but experimental adult fecundity was significantly less than control fecundity for the constant photoirradiation group.

KEY WORDS Photosensitization, photochemical, photoreactive, photoactive, photochemistry, photosensitizeq control

INTRODUCTION inconsequential when compared to the enormous damage caused when light is added (light reac- (PII) Photofrin@ porfimer sodium could be toxic tions). Because of this, all research has focused on photoirradiation, to adult without because it the light reactions and finding ways to make this larvae provided photoirradiation. was toxic to not activity cell specific (Dougherty 1987, Rebeiz The potential for light to pass through the scales of l99s). the adult body and activate the most destructive Porphyrins or hematoporphyrin derivatives pos- light reaction (with photoirradiation) pathways of sess alternating double bonds around 4 pyrrole PII would be minimal. The dark reactions (no pho- rings resulting in a conjugated I 8 rr electron system toirradiation) pathways would be the only reactions that provides high stability. The active component leading to cellular destruction in adults. Adults of PII seems to be a 2- to 3-unit oligomer that ab- might ingest PII in a sugar solution, because PII is sorbs light in the 400- to 630-nm range of the spec- reconstituted with 5Vo glucose, and sugar is provid- trum (Dougherty 1987, Byrne et al. 1990). This ed as a carbohydrate source for adults. Our focus oligomer, when activated by light in the presence was on the effect of PII on adult life span, egg of molecular oxygen, produces photoproducts de- production (fecundity), and fertility. structive to cells. Photosensitizers are compounds requiring direct Photodynamic effects of porphyrins are due to interaction with light and molecular oxygen to pro- their photoexcitable nature. The ground-state por- duce highly reactive oxygen species (primarily sin- phyrin is photoexcited glet oxygen). These compounds are toxic to all liv- to the singlet state. Next, ing things because singlet oxygen reacts with many intersystem crossing of an electron from the excited types of biological molecules (membrane lipids, cy- singlet state to the excited triplet state occurs. The tochromes, nucleic acids; Kessel 1981, 1986). Nat- excited triplet state has lower energy than the sin- ural and synthetic photosensitizers are equally de- glet state, but has a longer halflife. Now the ex- structive (Arnason et al. 1981. Sakurai and Heitz cited triplet sensitizer may undergo I of 2 reac- 1982, Kagan et at. 1987). tions-type I or II (Foote 1990). Indirect evidence One approach to photosensitizing cells involves indicates the primary pathway for cellular destruc- providing a natural or synthetic photosensitizer, tion is the type tr reaction. Tlpe II reactions occur such as PII, to living organisms or to target cells in when triplet sensitizer interacts with ground-state sufflcient quantities that when exposed to light and oxygen to produce excited singlet oxygen (Foote molecular oxygen phototoxic products are pro- l99O). Singlet oxygen is extremely reactive and duced and the organism is injured or killed. These will react with electron-rich substrates yielding per- are the light reactions, which are the most destruc- oxides and other oxidized species (Spikes 1985, tive to cells. Another approach involves use ofpho- Foote 1990). The effect of these oxidized species tosensitizer, such as PII, as stated above, but with- is disruption of metabolism, nucleic acids, and out exposure to or with little exposure to light. membranes (Foote 1990). Little inquiry has been initiated concerning this Barbieri (1928) demonstrated that aquatic larvae mode of PII action. Toxicity is known to be asso- of Anopheles maculipennis Meigen were killed ciated with the dark reactions. but this activitv is when exposed to halogenated fluorescein deriva-

248 Snrrnunrn2000 EFFEcrsor Psoropnlw@oN Epsru.|poonts 249 tives, erythrosin, and eosin and then illuminated. cage. Collected eggs (on egg papers) were a mix- Schildmacher (1950) confirmed the above work ture of autogenous and anautogenous eggs, were when he photosensitized An. maculipennis, Anoph- not stored, and hatched -48 h after oviposition in eles superpiuas Grassi, and Aedes aegypti (L.) 250-ml beakers or half-pint (279.3-rn1) Ball jars. mosquitoes with rose bengal, erythrosin, fluores- First-stage larvae were transferred to Nalgene@ cein, and eosin. Forty years later, Hartberg and Judy pans (VWR Scientific Products, Suwanee, GA) (32 (1990) showed that larvae of Eretmapodites quin- x 26 x 6 cm or 26 x 16 x 6 cm) half filled with quevittatus Theobald were killed by PII and that tap water (tHrO) and reared to tlre 4t}r larval or onset of effects was directly proportional to con- adult stages. During development larvae were pro- centration of Ptr and length of exposure to PII. Hel- vided :0.25 ml of larval food (Wardley'so fish leck and Hartberg (unpublished) also witnessed in- food and water,2;l ratio) placed at the bottom of creased difficulty in larval and pupal extrication, Nalgene pans. Experimental adults were provided but were unsure whether this was due to improper sugar cubes, and females were provided anesthe- chitin formation or reduced energy levels. For an tized mice for blood meals. The lst blood meal was excellent review of the history of insect photosen- provided when autogenous egg production ceased sitization see Heitz and Downum (1987). and subsequent blood meals were provided every This study examined the effects of Photofrin' II 2-3 days. All adults were photoirradiated with 8 or Photofrin@ porfimer sodium (PII) on the adult (15-W) Philips@ fluorescent lamps in parallel array mosquito Er. quinquevittatus. This species is native (1.12 mWcm', 350-800 nm), placed -25 cm to sub-Saharan Africa and Madagascar (Gillett above the container. 1972) and may transmit arboviruses (Macnamara Toxicity of photoirradiated PII on adults when 1953, Mclntosh et al. 1961, Gilotra and Shah 1967, fed PII as 4th-stage larvae: Fifty 4th-stage larvae Serie et al. 1968. Brottes et al. 1969). (-6 days of larval development) each were fed lar- Eretmapodites quinquevittarur was chosen as the val food and 3.75 pg/ml of PfI (reconstituted with test organism because it is easily maintained in the dextrose), and were cultured in I of 3 different os- laboratory and has been studied extensively (Hart- motic solutions until emergence of adults: hypoton- berg and Gerberg 1971; Hartberg and Johnston ic 1: 6i.,ttt d water), hypertonic (: 1.1x Ae. ae- 1977,1979; Johnston and Hartberg 1981; Hartberg gypti saline solution), and isotonic (: Ae. aegypti and Faircloth 1983; Helleck et al. 1993, 1994. saline solution) (Hayes 1953). These 3 osmotic so- 1996, 1997). lutions were used because we found that different This research was conducted to further assess the osmotic conditions coupled with the effects of PII efficacy of photosensitizers as control agents have different effects on larval survivorship (Hel- against adult mosquitoes. Resistance to and lack of leck et al. 1997). Additionally, 50 larvae were giv- effectiveness of insecticides by mosquitoes occurs en larval food and either cultured in a hypertonic and is life-threatening, because mosquitoes are solution containing no PII (control 1) or tHrO con- known vectors of disease (Georghiu and Lagunes- taining no PII (control 2) until emergence of adults. Tejeda 1991, Raymond et al. 1993, Pasteur and Fourth-stage larvae fed PfI that developed into Raymond 1996). Photoactive compounds could adults (parentals) were mated with control 2 adults. provide effective photoinsecticides for future use in Resulting F, adult siblings were interbred. Adult insect control (Spikes 1985, Rebeiz et al. 1995). life span, fecundity, and fertility were determined and analyzed for these 2 generations of adults. Toxicity of nonphotoirradiated PII on adults: MATERIALS AND METHODS Ttventy-five adult (-12-13 days of development) Laboratory colonies and cultures of Er. quin- males and 25 adult females each were randomly quevittatus (EQ-MIXED) were maintained at the placed in 6 l-gallon (3.8-liter) cages. Three of the Medical Entomology Research Laboratory at Bay- cages (experimentals) included a 50-ml beaker con- lor University in a walk-in environmental chamber taining 90 pglml of PII (reconstituted with dex- set at 25"C, 8O + 5Vo relative humidity, and a 14: trose; 20-ml final volume). The other 3 cages (con- 10 h light: dark photoperiod. The EQ-MIXED adult trols) included a 50-ml beaker contnning a 5Vo colony was provided sugar cubes as a carbohydrate dextrose solution (20-ml flnal volume), but no PII. source, and females were provided anesthetized One experimental and I control cage were placed mice every 2-3 days for blood meals. This colony in complete darkness, 1 experimental and I control was established by W. K. Hartberg from larvae col- cage were placed under constant photoirradiation lected from a tree hole in the l(isutu section of Dar (: l.lZ mWcm2, 350-800 nm; positioned 25 cm es Salaam, Tanzaria, in July 1969 (Hartberg and above the container), and 1 experimental and I con- Gerberg 1971). trol cage were placed under intermittent photoirra- Eggs were collected from the EQ-MD(ED colo- diation (: environmental chamber light, 14:10 h ny by providing ovipositing females with moist, photoperiod). bleached, paper towels (oviposition or egg papers) Statistical analysis: Life spans of parentals were lining the inside of 250-ml beakers or half-pint compared to those of F, adults by the log-rank or (279.3-ntl) 3u11o jars placed inside of the colony chi-square test (Anderson et al. l98O). Fecundity of 250 JoURNAL or ruB Alras,nrcervMoseurro CoNrnol AssocrerroN Vor-. 16. No. 3

Table l. Adult life span in days, fecundity (eggs laid), and fertility (percent hatch) for autogenous (auto) and anautogenous (anauto) eggs of Eretmapodites quinquevittatus larvae (3rd/4th stage) developing into adults after being exposed to 3.75 pg/ml of Photofrin porfimer sodium II and hypotonic or isotonic solution. Experimental parental (P,) adults were crossed with parental controls of opposite sex. Standard enors are in parentheses.r

Experi- ment Pl Life span (days) Eggs laid Eggs hatched Percent hatch no. MXF (M/F) (auto/anauto) (auto/anauto) (auto/anauto) I Cont x Hypo 9157 ot25 ot20 0/80 2 Iso x Cont 2t9 ot3 o/2 ot66.6 J Iso X Cont t/57 0/0 0/0 0/0 4 Cont X Hypo 27t57 ot29 0/0 0/0 5 Cont X Hypo 18t54 on4 otl ot7 6 Cont X Hypo 7t& ot26 ot20 on7 7 Cont X Hypo tU35 on2 0/0 0/0 8 Cont x Hypo ty64 o/3 oto oto 9 Cont x Hypo Batch 1 24/44 o/28 o/27 o/96 9 Cont x Hypo Batch 2 Same ol63 o/37 ot59 Mean (average) r2.2 (3.O)/ 25.3(10.0) r3.3(7.9) 52.7 49.0(5.8)

'M, male; f; female; Cont, control; Hypo, hypotonic; Iso, isotonic. parentalswas comparedto that of Fr adults by com- motic concentrations are fully discussed in Helleck parison of average eggs laid/female. Fertility of et al. (1997). Experimental parentals did not differ parentals and Fr adults were compared by l-way signiflcantly in life span when comparedto control analysis of variance (ANOVA) (arcsine transfor- I parentals (P > 0.05). None of the experimental mation). parental females laid autogenous eggs but did lay Adult life span, fertility, and fecundity were de- 203 anautogenouseggs for a mean of 25.3 (SE : termined for each l-gallon (3.8-liter) cage. Adult 10.0) anautogenouseggs/female (Table 1). Fertility life span of experimentals was compared to that of for these eggs was 52.7Vo.The F, adults lived sig- controls by the log-rank or chi-square test (Ander- nificantly longer than parentals(P > 0.05; Thble 2). son et al. 1980). Fecundity of experimental adults Each F, female laid an average of 26.1 autogenous was comparedto that of controls by comparison of and 200.5 anautogenouseggs (Table 2). Fertility for average eggs laid/female. Fertility of experimental eggs laid by parental females (52.7Vo)was signifi- and control adults was compared by 1-way ANO- cantly less than fertility of eggs laid by F, females VA (arcsine transformation). (94Vo autogenous and 82.7Vo anattogenous; P ( 0.05; Table 2). All control 1 and control 2 4th-stage larvae developedinto adults. RESULTS Toxicity of photoirradiated PII on adults when fed PII as 4th-stage larvae Todcity of nonphotoirradiated PII on adults Ttvo isotonic and 7 hypotonic 4th-stage larvae Survival rates of experimental adults fed PII (90 developed into parental adults and were mated with pglml) as adults with no photoirradiation, constant control I parentals of the opposite sex (Table l). photoirradiation, or intermittent photoiradiation None of the hypertonic males or females survived did not significantly differ for males or females (P to the adult stage. The effect of differences in os- > 0.05; Figs. I and 2). Survival rates of experi-

Table 2. Mean life span in days, fecundity (eggs laid), and fertility (percent hatch) of autogenous (auto) and anautogenous (anauto) eggs laid by lst. generation (F,) females of Eretmapodites quinquevittat&s. First-generation adults were interbred. Standard errors are in parentheses.l

Mean life Experiment Fl span (d) Eggs laid Eggs hatched Percent hatch no. MXF (M/F) (auto/anauto) (auto/anauto) (auto/anauto) I 9MX9F 52.8(9.0)182.8(14.4) 29712,640 28612,461 96193 2 IM 86.0(0.0)/0 0/0 Oto 0/0 6 6MX5F 39.6(8.5)/s8.8(r6.3) 63t295 621212 98172 9 5Mx13E,Batchl 37.4(2.7)/72.7(13.6) 35012,948 32712,042 93169 9 5Mxl0EBatch2 50.8(11.2y38.8(10.3) 25611,536 23411,422 91192 Mean (average) 47.7(4.rteJ1.2) 26.U2OO.5 24.5116s.8 94182.7

'M, malel E female Spprelaaen 20OO 251

I 0.e h 0.9 -----l- Non_ Photo. b -*-- Non_Photo. e o.8 €. o.t -Non-Photo. 3 o.z -Non-Photo. 3 o.z Control Control € o.e € o.o +lnter. Phot. O n < +Inter. Photo. g o.5 ------

mental adults were significantly lower than for con- colonized females of Er. quinquevittatus averaged ( trol adults in all 3 photoi:radiation settings (P 137.1-244.8 anautogenous eggs/female, whereas 0.001). Fecundity was lowest for experimental fe- the 8 females of this experiment laid an average of males exposed to PII in the constant photoirradia- 25.3 eggslfemale(Table l). Anautogenousegg fer- tion group and fertility did not differ (Table 3). tility (percent hatch) was also reduced. Helleck et al. (1996) reported fertility of 87.8 and 74.5Vofor DISCUSSION autogenous and anautogenous eggs, respectively. Females from this experiment laid anautogenous Toxicity of photoirradiated PII on adults when eggs that hatched at a 53Vo rate (Table 1). First- fed PII as 4th-stage larvae generation females laid an average of 24.5 and, The importance of this line of investigation is 165.8 autogenousand anautogenouseggs, respec- evident when realizing that even lower concentra- tively (Table 2). Although autogenousegg produc- tions of PII (1-5 pglrrl, sublethal) might not kill tion was less than previously reported, anautoge- larvae (or other nontarget organisms), yet might ad- nous egg production was consistent with that of versely affect developing adults or later generations previous reports (Helleck et al. 1996). Fertility lev- (Table 1). Although PII fed at I stage can advers€ly els of 94Vo and.83Vofor autogenousand anautog- effect subsequent stages of development, it was not enous eggs were consistent with previous reports as "Iable clear whether Ptr fed during 1 generation could ad- well (Helleck et al. 1996; 2). We did not de- versely effect a subsequent generation. tect PII fed to I generation having any effect on Although survivorship of experimental parentals subsequentgenerations. did not significantly differ (P > 0.05) when com- pared to their control 1 mates, tiey were affected Toxicity of nonphotoirradiated PII on adults in other ways. None of the parental females laid autogenous eggs, which differs geatly from a range Adults of Er. quinquevittatus are affected by Ptr. of 53.3-64.5 autogenous eggs/female that is re- As was initially hypothesized, the dark reactions ported for colonized Er. quinquevittatus females were most important in killing adults that are fed (Helleck et al. 1996). Anautogenous fecundity was PII. This is a plausible assumption,because light also reduced. Helleck et d. (1996) reported that should not easily penetrate the scales of an adult

Table 3. Fecundity and fertility of adult females of Eretmapodites quinquevittatzs exposed to 9O mg/ml of Photofrin porfimer sodium (PII).l

Intermittent Constant Non-photoirradiation photoirradiation photoirradiation Ptr Control Control PII Control No. eggs laid 1,249 r,642 1,207 r,533 702 I,158 No. eggs hatched I,ll1 t,445 1,038 r,380 667 r,054 Percent hatch 89 88 86 90 95 9l 'Nonphotiradiation, PII with no photoinadiation; intemittent photoinadiation, PII with 14:10 h light:dark photoinadiation cycle; constant photoinadiation, PII with constant photoinadiation; controls, no PII with same photoinadiation.

L 252 JounNel or nrn Altenrcel Moseurro Corrrnol AssocnnoN Vor-. 16, No. 3 mosquito and because different exposures or inten- Hartberg WK, Johnston KW. 1977. Red-eye, a sex-linked sities of light had no significant affect on the sur- mutant in the mosquito Eretmnpodites quinquevittatus vival rate of adults fed the same concentration of Theobald. Mosq News 37:725--728. Ptr. Control adults survived significantly longer Hartberg WK, Johnston KW. 1979. White-eye, a mutant in the mosquito Eretmapodites quinquevittatzs Theo- than experimental adults in all 3 light settings (Figs. bald. Mosq News 39:371-373. I and 2). Adults living in constant photoirradiation Hartberg WK, Judy MM. 1990. Photosensitization of mos- laid significantly fewer eggs than controls in con- quito larvae with DHE. In: Proceedings of the 1990 stant photoiradiation or any of the other experi- South Central Regional Mosquito Control Conference mental groups (P < 0.05; Table 3). Fertility was 34th Annual Meeting. p 22. not different among any of the experimental or con- Hayes RO. 1953. Determination of a physiological saline trol groups. The females in constant photoirradia- solution for Aedes aegypti. J Econ Entomol 46:624- tion may have laid fewer eggs because of reduced 627. energy levels caused by reduced energy or food ab- Heitz JR, Downum KR, eds. 1987. Light-activated pesti- sorption by damaged gastric epithelia (Helleck and cides. ACS Symposium Series. Washington, DC: Amer- ican Chemical Society. Hartberg 1999) due to constant photoirradiation Helleck AM, Hartberg WK. 1999. Site of Photofrin IIo their entire lives. photosensitization in larvae of Eretmapodites quinquev- Adult life span and fecundity were significantly ittatus T-heobald. J Am Mosq Control Assoc 15:.437- reduced when adults ingested PII. A solid form of 445. photosensitizer and carbohydrate placed in areas Helleck AM, Hartberg WK, Baldridge RS. 1997. Photo- (hanging under eaves of the home, and so on) sensitization of Eretmapodites quinquevittatus Theo- where adult mosquitoes feed or rest might be useful bald (Diptera: Culicidae) eggs and larvae with Photof- in reducing the number of females or in reducing a rin II@. "/ Am Mosq Control Assoc 13:292-295. female mosquito's ability to lay eggs. Helleck AM, Hartberg WK, Vodopich D. 1993. Daily sur- vivorship and life span of the mosquito Eretmapodites quinquevittatus Theobald (Diptera: Culicidae) under REFERENCES CITED laboratory conditions comparedto Aedes aegypti, Aedes albopictus, and Aedes bahamensis. Bull Soc Vector Anderson S, Auquier A, Hauck WW, Oakes D, Vandaele Ecol 18:109-113. W, WeisbergHI. 1980. Statistical methods cornpar- for Helleck AM, Hartberg WK, Vodopich D. 1994. Variations ative studies: techniques bias reduction New York: for in development times of Eretmapodites quinquevittatus John Wiley & Sons. Theobald (Diptera: Culicidae). 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