Journal of the AmericanMosquito Contol Association,19(2):155-158, 2003 Copyright @ 2003 by the American Control Association, lnc.

PH ORM I D I UM AN I M ALIS (CYANOB ACTERIA : OSCILLATORIACEAE) SUPPORTSLARVAL DEVELOPMENT OF ALBIMANUS

M. GUADALUPE VAZQUEZ-MARTiNEZ,I MARIO H. RODRiGUEZ,' JUAN I. ARREDONDO-JIMENEZ'AND JOS6 D. MENDEZ-SANCHEZ,

ABSTRACT, The capability of Phormidium animalis, a cyanobacterium commonly found in larval habitats of Anopheles albimanus in southern Mexico, to support larval development of this mosquito was investigated. First-stage larvae were reared under insectary conditions wilh P. animali^r ad libitum and their development was compared with larvae fed with wheat germ. The time of pupation and adult mosquito size, assessed by wing length, were similar in both groups, but fewer adult mosquitoes were obtained from larvae fed with the cya- nobacteria. Nevertheless, these observations indicate that P. animalls is ingested and assimilated by larval An. albimanus, making this cyanobacterium a good candidate for genetic engineering for the introduction of mos- quitocidal toxins for malaria control in the region.

KEY WORDS Anopheles albimanus, Phormidium animalis, Chlorella cf . sorokiniana, mosquito larvae, feed- ing ecology, , Mexico

INTRODUCTION in southern M6xico (Rodriguez and Loyola 1989). Field studies conducted on a portion of this coastal Studies on mosquito larval feeding behavior in- plain indicated that 19 species of cyanobacteria in- dicate that selection of food particles is largely de- habited larval habitats of An. albimanus (Y6zqtez- termined by particle size. Only those particles that Martinez 1997), but only 6 species could be isolat- fit the mouths of larvae are ingested (Wallace and ed from larval midguts of local An. albimanus. The Merritt 1980, Ali 1990, Thiery et al. 1991) at the majority of these species were from the most com- same proportion as the particles are present in the monly encountered cyanobacteria in water samples. breeding site water (Khawaled et al. 1989, Ydz- Because Phormidium animalis (Agardh. ex Go- quez-Martinez 199'7). However, among ingested mont) Anagnostidis and Komarek (Cyanobacteria: food particles, some could be more nutritious or Oscillatoriaceae) was abundant in larval habitats more easily digested than others (Thiery et al. but could not be isolated from larval midguts, it 1991, Sangthongpitag et al. 1996). This fact is par- was assumed to be readily digested after its inges- ticularly important in the search for alternative ap- tion (V6zquez-Martinez et al. 2OO2).The objectives proaches in malaria vector control, where the pos- of the present study were to isolate and grow P. sibility of using genetically engineered natural animalis in culture, to confirm that P. animalis is foods to control mosquito larvae currently is being readily ingested and digested by larval An. albi- explored (Liu et al. 1996, Porter 1996). manus, and to assess the importance of P. animalis Aquatic cyanobacteria, widely distributed organ- as a nutrient for mosquito larvae by verifying the isms in mosquito larval habitats, are good candi- ability of this species to support the full develop- dates for genetic modification because they can ment of larvae to adulthood. withstand severe fluctuations of water chemistry (Sangthongpitag et al. 1996, Y{zquez-Mart(nez 1997), they are ingested and digested by mosquito MATERIALS AND METHODS larvae (Merritt et al. 1992), and they have relatively simple genomes. Previous attempts to genetically Cyanobacterial isolation from water samples of engineer cyanobacteria with mosquitocidal toxins larval habitatr.' Cyanobacteria were maintained in (Tandeau de Marsac et al. 1987, Angsuthanasombat petri dishes (100 X 15 mm) in a laboratory at 26 -r and Panyim 1989, Wu and Federici 1993, Geor- 2"C and 1O-8OVo relative humidity. Artificial ghiou and Wirth 1997, Xiaoqiang et al. 1997) used light was provided from OSOOto l53O h and the laboratory colonies of cyanobacteria that may not remaining light available was daylight passing be natural food of mosquito larvae, and thus, may through the windows. Cyanobacteria were cultured not be able to colonize natural larval habitats. in fresh BG-I1 medium once every 3 months. Anopheles albimanus Wiedemann is the main Phormidium animalis was isolated from larval vector of malaria along the coastal plain of Chiapas habitat water samples as described by Ydzqtez- Martinez et al. (2002). Briefly, samples stored in the dark for 24 h at room temperature were inoc- rCentro de Investigaci6n de Paludismo, Instituto Na- ulated into liquid BG-l1 medium (10 g agaa 10 g cional de Salud Priblica, Apartado Postal 537, Tapachula, Chiapas, M6xico. NaCl, 1.5 g NaNOr, MgSO..TH,O) and incubated 2 Centro de Investigaciones Sobre Enfermedades Infec- under fluorescent light (Thiery et al. 1991). After 7 ciosas, Instituto Nacional de Salud Pfblica, Avenida days, samples were inoculated into BG-l1 medium Universidad No. 655, Colonia Santa Marfa Ahuacatitl6n. solidified with lVo Bacto-Agar@ (Bioxon, Mdxico Cuenavaca, Morelos 62508, M6xico. City, Distrito Federal, M6xico) (Rippka et al. 1979),

155 t56 JounNaL op rna AuBnrcAN Moseurro CoNrnol Assoctn.r.rtN VoL. 19, No. 2 and further exposed to a fluorescent light. Cyano- into emergence chambers (30 X 30 X 30-cm alu- bacterial growth was examined daily and positive minum-screened cages). The number of emerging cultures were purified by serial dilutions in both adult mosquitoes was recorded for each diet to ob- solid and liquid BG-ll media. Cyanobacteria were tain the emergence success. The mean proportion identifled with standard keys (Desikachary 1959, of adult mosquitoes emerged from larvae reared un- Prescott 1970, Anagnostidis and Komarek 1985, der both diets were conlpared with unpaired 2- Knutson and Sterk 1996). tailed r-tests of arcsin V2-transformed data (Zar Purified cyanobacterial cultures: Samples of fil- 1999). aments of P. animalis were cultured in BG-l1 liq- Body size of adult mosquitoes: The size of adult uid medium for 8 h to enrich the cyanobacterial An. albimanus was estimated by the length of their population and then inoculated on plates of BG-l I wings. The mean wing length of mosquitoes was medium solidified with l%o Bacto-Agar. Cultures measured from the axillary incision to the outer + were incubated at26 2oC in a 12:12 h lisht:dark margin of the Rl vein (Xue and Ali 1994). Mea- photoperiod, with two 39-W white fluoresc-ent light surements of mosquitoes reared with P. animalis or lamps situated 73 cm from the plates. The cultures the control diet were compared by sex with un- were washed and centrifuged 6 times at 2,000 X g paired 2-tailed t-tests (Zar 1999). for 60 sec. Filaments were resuspended in lO0 ml of BG-11 liquid medium containing penicillin G at RESULTS 30 pglml and streptomycin at 50 pglmt. The purity of the algae was assessed by microscopic obser- Digestion of cyanobacteria vation in a phase-contrast microscope (Leitz Dialux Most filaments of P. animalis lost their emerald- 22@,Leica Microsystems, Wetzlar, Germany). green coloration and became a brownish and pale Digestion of cyanobacteria.. Tests were conduct- yellow color in larval guts obtained 3O min after ed to assess if ingested P. animalis were digested feeding. No cyanobacterial growth was observed by larval An. albimanus (white-stripe strain; Chan when the gut contents were cultured on BG-I1 me- et al. 1994). Filaments of P. animalis were offered dium. At the same time, whole undamaged C. cf. in filtered water for 30 min to 4th-stage mosquito sorokiniana were observed in larval gut contents, larvae that had been starved overnight. The gut and cyanobacterial growth was observed in cul- contents of 3 larvae were examined under a light tures. microscope and the gut contents ofanother 3 larvae Emerald-green filaments of P. animalis were ob- were cultured in BG-l1 medium. A control larval served in samples of larval gut