Cyclopoida: Cyclopidae) As Biological Control Tools for the Dengue Vector Aedes Aegypti

Home , Cyclopidae, Macrocyclops, Macrocyclops albidus

Joumal of the American Mosquito Control Association, l3(l):18-23, 1997 Copyright A 1997 by the American Mosquito Control Association, Inc.

EVALUATION OF CARIBBEAN STRAINS OF MACROCYCLOPS AND MESOCYCLOPS(CYCLOPOIDA: CYCLOPIDAE) AS BIOLOGICAL CONTROL TOOLS FOR THE DENGUE VECTOR AEDES AEGYPTI

S. C. RAWLINS, R. MARTINEZ, S. WILTSHIRE, D. CLARKE, P PRABHAKAR AND M. SPINKS

Caribbean Epidemiology Centre (CAREC), P. O. Box 164, Port-of-Spain, Trinidad

ABSTRACT. Fifteen Caribbean strains of copepods were assessed for their predation ability against mosquito larvae. Macrocyclops albidus from Nariva, Mesocyclops aspericornis from Oropouche, and Mesocyclops longisetus from El Socorro, Trinidad, were most effective against Aedes aegypti but not against Culex quinquefasciatus. Mesocyclops longisetus and Me. aspericornis prevented any mosquito survival over 25 wk of observation despite weekly challenges with Ae. aegypti. The copepods were tolerant to dosages of the insecticide temephos that are usually toxic to mosquito larvae. This indicated that copepods could be incorporated into an integrated control system. To determine whether pathogenic microbes might be introduced with copepods into drinking water, microbial studies were done on the copepods. These showed the presence of only Aeromonas .sobria, Pseudomonas sp., Alcalignes sp., and gram-positive bacilli. Although none of these are highly pathogenic to humans, the application of these copepods has not yet been recommended for use in drinking water.

INTRODUCTION 1992). ln contrast, the larger M. longisetu,s killed IOOVo of Ae. aegypti, Anopheles farauti Laveran, The continuing endemicity of dengue serotypes ard Culex quinquefascialas Say. 1, 2, and 4 in the Caribbean region together with Vasconcelos et al. (L992) in small-scale field tri- the risk of introduction of dengue type 3 from Cen- als in Brazil found that M. longisetus controlled Ae. tral Arnerica has greatly emphasized the need for aegypti in containers that produced more than 8O7o efficient managementof the vector Aedes aegypti of the mosquito. They found the involvement of the (Linnaeus)(Nathan 1993, Pan American Health Or- community in such a project to be very helpful. In ganization 1994). There is evidence of failure of Mexico, M. longisetus selected from 15 species of source reduction practices for controlling this con- copepods collected in Nuevo Leon and Coahila was tainer breederdue to the reluctanceof some Carib- equally predaceous on both Ae. aegypti and Cx. bean householdersto discard most potential mos- quinquefasciaras (Rodriguez 1992). In the Carib- quito-producing containers (Rosenbaum et al. bean islands of Puerto Rico and Anguilla, M. as- 1995). Also, the development of insecticide resis- pericornis controlled but did not eliminate Ae. ae- tance in several Caribbean populations of Ae. aegypti in drum habitats (Suarez 1992). gypti (Rawlins and Hing Wan 1995) has forced The prospect of incorporating copepods into an mosquito control authoritiesto seek alternative sus- integrated control system for mosquitoes has at- tainable systemsfor managementof this mosquito. tracted the attention of several mosquito control Biological control tools such as the mosquito workers. Thus, the combination of copepods with Toxorhynchitesmoctezuma (Dyar and Knab) have Bacillus thuringiensis var israelensis (B.t.i.), Ba- been evaluated for management of Ae. aegypti cillus sphaericrrs, and methoprene (Tietze et al. (Rawlinset al. 1991,Tikasingh 1992). This hasmet 1994), or with insecticides (Marten et al. 1993), has with only minimal success due to failure of the improved the performance of various copepod spe- predator to flnd and colonize habitats of its prey in cies. urban environmentswhere Ae. aegypti is known to This paper evaluates the larval consumption thrive. characteristics of a number of Caribbean copepod Recently, certain cyclopoid copepodshave prov- taxa and their capability of surviving in insecticide- en to be capable of controlling Ae. aegypti larvae treated habitats and their potential for incorporation in peridomestic breeding containers (Marten 1990, into an integrated control program for Ae. aegypti. Marten et al. 1994). Marten et ^1. (1992) reported The present study provides data from such work that one species, Mesocyclops longisetus (Thi€' performed in Port-of-Spain, Trinidad. baud), was capable of reducing Ae. aegypti larval populations by more thar99.9Vo. MATERIALS AND METHODS Other authors have evaluated some Nonh Amer- ican copepods against container-breedingmosqui- Collection of copepods: Various copepod sam- toes such as Aedes albopictus (Skuse)(Schreiber et ples were collected from freshwater bodies in dif- d. 1993). Brazilian strains of Mesocyclopsasperi- ferent Caribbean locations. The copepods were cornis (Daday) showed Potential as biological con- taken to the entomological laboratories at the Ca- trol agents against Ae. aegypti but were not as ef- ribbean Epidemiology Centre (CAREC; Port-of- fective against Anopheles and Culex (Kay et. al. Spain, Trinidad) and were reared on a diet of Par-

l8 MrncH 1997 Copppoos Ls ,q.BtocoNrnoI- TooL t9 amecium caudatum, Chilomonas sp. (Suarez et al. of these 100 lst-instar Ae. aegypti larvae were add- 1992), artd lst-instar mosquito larvae. For each ed and the drums were covered with a fine mesh to sample a culture was started from a single gravid prevent entrance of any gravid mosquitoes. The female. The copepods were maintained under lab- drums were left under partially shadedconditions. oratory conditions at ambient temperatures of 2t- At 6-day intervals, all larvae and/or pupae were 34'C until they were evaluated for their predatory removed from each drum by very c:reful sweeping ability against lst-instar mosquito larvae. Copepod with a fine nylon dip net and emptied into enamel samples from each strain were sent to Janet Reid trays. Larvae and/or pupae present were counted, at the Smithsonian Institution, Washington, DC, removed, recorded, and the water containing the who kindly verified the species identifications. A copepods and/or temephos was returned to its re- strain of M. longisetus curvatus (Dussart) was also spective drum. A new set of 100 lst-instar Ae. ae- received from M. E. Suarez. CDC. San Juan Lab- gypti larvae was added to each drum and surviving oratories, Puerto Rico, and was used as a reference larvae, if any, were harvested after 6 days. This predatory strain. processwas continued for 25 wk. Predation studies: First-instar Ae. aegypti larvae Field studies in tire habitats: Discarded auto- were introduced into each well of a tissue culture mobile tires were selectedfor this field study in St. plate each containing one adult copepod in l0 ml James,Port-of-Spain, Trinidad, with a known pres- of water. Six replicates were done for each strain/ ence of both Ae. aegypti and Cx. quinquefasciatus- species. After 24 h, the percent kill was assessed Tires were each treated with one of the following by removing and counting the number of live and regimes: 1) tire 1, IOOM. longisetus in 2 liters of dead larvae. and a new set of lst-instar larvae was water; 2) t$e 2, 20O M. longisetus in 2 liters of exposed to the copepods. This process of evaluation water; 3) tire 3, 10OM. aspericornis in 2 liters of was repeated 5 times and the mean percent mortal- water; 4) ttre 4, 2OOM. aspericorrtis in 2liters of ity of Ae. aegypti larvae for each copepod strain/ water, and 5) tire 5, untreated control, 2 liters of species was assessed. water. Insecticide susceptibility of copepods: Prelimi- Nine different sites were used for each treatment. nary laboratory testing was conducted on 4 strains The tires were left in shaded locations without pro- of copepods: Macrocyclops albidus principalis tection from gravid wild mosquitoesfor 14 wk. The (Herbst) (Nariva), M. longisetus (CDC), M. Iongi- water in the tires was examined by emptying the serus (El Socorro), and, M. aspericornis (Daday) contents into a white enamel bowl and any mos- (Oropouche). From each strain, l0 copepods were quito larvae present were removed with a pipette placed into their respective wells of a tissue culture and taken to the laboratory. This was done at4-day plate with 10 ml of solution containing varying intervals becauselonger periods could permit pu- concentrations of temephos. The 6 concentrations pation and a possible risk of releaseof adult mos- chosen ranged from 0.02 mg/liter (the diagnostic quitoes. The uncounted copepods were returned to dosage for Ae. aegypti larvae) to O.22 mg/liter. their respective tires and the water brought back to Mortality was determined by the presence of mor- its original level. In the laboratory, the larvae for ibund or dead copepods at 24-h intervals over a each mosquito specieswere counted and recorded 2-wk period. No food was offered to the copepods for each tire habitat. in both the controls and test wells during this pe- Statistical treatment: The data were entered us- riod. ing the EPI-Info version 6 program provided by the Copepod/temephos integrated control trials: CDC of the U.S. Public Health Service (Division Two of the more predaceous species of copepods, of Surveillance and Epidemiologic Studies, Epide- Mesocyclops sp. B (near aspericornis) from Chag- miology Program Office, CDC, Atlanta, GA). A re- uaramas and M. aspericornis from North Oropou- peated multivariate analysis of variance was used che, Trinidad, were selected for this tial. Mesocy- to compare regimes, with weeks being selectedas clops longisetus curvatus obtained from CDC in the repeated factor. Verification of the assumptions Puerto Rico was used for comparison to the 2 Trin- for these statistical tests was made through residual idad strains of copepods. analysis. In caseswhere the assumptionswere not TWenty-four drums each containing 200 liters of satisfied, a square root transformation was utilized tap water were allowed to stand covered for 7 days and the test repeated.The usual F statistic was the before any additions were made to tlem. For each criterion applied for assessmentof group compari- strain of copepod, drums were prepared as follows: sons. Unless otherwise mentioned, the P value of l) no treatment, controls (3 drums); 2) 8 g of 5Vo was used as the determinant level of signifi- Abate@ (Cyanamid Intl., Wayne, NJ) l7o sand gran- cance.The STAT module of the StatisticalAnalysis ules (0.4 g temephos) (3 drums); 3) 100 adult co- Software (SAS) Version 6 for Windows (SAS In- pepods alone (9 drums) (3 drums per species); and stitute, Inc., SAS Campus Drive, Cary, NC) was 4) lOO adult copepods plus 8 g of Abate l7o sand used for analyzing the data. granules (0.4 g temephos) (9 drums) (3 drums per Bacteriological analysis of copepods: Bacterio- species). logical assayswere conducted on 2 speciesof co- There were a total of 24 drum habitats. To each pepods to determine whether there was a risk of 20 JounN,ct- oF THE AMERICAN Moseurro CoNrnol AssocrlnoN VoL. 13, No. I

Table l. Selective predation of copepods (Macrocyclops and,Mesocyclop.s) on l-instar Aedes aegypti and Culex q uinq uefasc iatas larvae. I

Mean Vo predation Taxa Aedes Culex Mac rocyclops albidus (Nariva) 85.7 63.1 Mesocyclops aspericornis (Oropouche) 83.5 J5- I Mesocyclops longisetus (El Socorro) 78.1 58.9 M es ocyc lop s near aspe ric o rnrs (Chaguaramas) 75.7 45.7 Me socyclops e llipticus (BBF) 79.O 41.6 Mesocyclops sp. (Arena) 77.8 42.8 Mesocyclops near longisetus (AWF) 59-7 46.4 M esocy c lop s lon gi setus (CDC) 72.5 43.1 M. aspericornis (Dominica) 73.9 37.4 Mesocyclops sp. (Antigua) 7l.l 34.7 Mesocyclops sp. (Mucurapo ground pool) 45.8 32.O Mesocyclops sp. (Mucurapo stream pool) 2t.2 lo.6 Mesocyclops sp. (Otoire) 7.4 3.8

' Controf mortalities were: Aedes alone, OVo: Aedes and Culex, O.6Vo and 5.47o, respectively; Culex alone, 5.7Vo introducing pathogenic bacteria into water storage erally, predation against Cx. quinquefasciatus was containers with the copepods. The copepods, M. of a lower order by all the copepod strains/species, longisetus arrd M. aspericornis, from CAREC in- ranging from 3.8 to 63.lVo. door and outdoor cultures were strained and about The 4 strains of copeods showed markedly dif- 30 individuals from each group were homogenized ferent insecticide susceptibilities to the larvicide te- in sterile containers. The homogenates and water mephos. The results were M. albidus (Nariva) 67o samples from these 2 cultures were analyzed for (lowest mortality) < M. longiseras (CDC) l2%o < bacterial agents by the methods described in Len- M. aspericornis (Oropouche) 47.3Vo < < M. lon- nette (1985). gisetus (El Socorro) 73.l%o (htghest mortality). The comparisons of larval Ae. aegypti survival in drums treated with the insecticide temephos, co- RESULTS pepods, temephos and copepods combined, or un- Copeod taxa collected in Caribbean countries: treated controls are shown in Fig. 1. For the cope- Some 15 taxa of copeopds were collected from var- pod Mesocyclops sp. near aspericonls (Fig. lA) ious Caribbean teritories. These consisted of 3 the copepod alone demonstrated control of Ae. ae- strains of Mesocyclops sp. A (near ellipticus) from gypti.Between weeks 3 and 18, containers with co- Tobago and Trinidad, artd Mesocyclops sp. B (near pepods alone kept the mosquito production between aspericornis) and sp. C (near longisetus) from Trin- 2 and l87o of the weekly introduced larvae. idad. There were 4 samples of M. aspericontis from Copepods of the M. aspericontis (Oropouche) Trinidad and Dominica, one sample of M. longi- and M. longisetus (CDC) strains maintained their setus curvatus from El Socorro. Trinidad. and M. mosquito larval population at OVosurvival through- albidus principalis from Nariva Swamp, Tiinidad. out the 25 wk of the study (Figs. 18, 1C). Control In addition, there was one sample each of Para- (untreated) drums were constantly productive, pro- cyclops chiltoni (Thompson), Paracyclops sp. A, ducing between 95 and 98Vo of weekly introduced Elaphoidella sp. A from Trinidad, and Allocyclops (lst-instar) larvae. Drums treated with temephos sp. from Tobago. alone were free of mosquitoes initially but began The comparative predatory ability of the 13 to lose their efficacy after week 17, and by week strains of copepods against lst-instar larvae of Ae. 25 were as productive as the control containers. aegypti and Cx. quinquefasciatus is shown in Table Drums treated with M. aspericornis (Oropouche) l. Macrocyclops albidus collected from Nariva plus temephos and M. longisetus (CDC) plus te- Swamp, Trinidad, was the most predaceous strain mephos were both very effective against Ae. ae- against Ae. aegypti (85.7Vo). Mesocyclops asperi- gypti prodtction (Figs. 18, lC), but Mesocyclops cornis from North Oropouche (83.57o) and M. lon- sp. near aspericornis was effective to a lesser de- gisetus from El Socorro, Ttinidad, (78.l%o) were gree (Fig. 1A). At the end of the 25-wk study, co- also very strong predators, comparing well with the pepods were present in all drums into which they reference M. longisetus CDC srain (72.5Vo). Gen- had been introduced, but they were not quantified.

Fig. 1. Weekly survival of Aedes aegypti larvae in drums treated with copepods and/or temephos. A. Mesocyclops near aspertcornis, B, Mesocyclops aspericornis (Oropouche strain). C. Mesocyclops longisetosus. M,qncs 1997 CoPEPoDsns l BtocoNrnor- Toot- A

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ers, for example, Vasconcelos et al. (1992) in Brazil lAedes ae€UpliEcdex quingJsleEelsus and Rodriguez (1992) in Mexico, who assayed Me- socyclops and found them to be efficacious as a E biocontrol tool against Ae. aegypti. :80 o Macrocyclops albidus (Cocal), M. aspericornis CI (t (Oropouche), and the reference species M. longi- Qeo L seras exhibited tolerance to the organophosphorous -g insecticide temephos. This tolerance suggests the o40 o- validity of incorporating insecticide-tolerant cope- = pods as part of an integrated control system for Ae. oegypti: accidental or intended insecticide contam- ination will not necessarily eliminate the copepod population. Also, a complete change over from te- 1 2 4 5 mephos to copepod biocontrol would not present ,,". &ou" many problems. These strains of copepods, by not Fig. 2. Mosquito larvae found in tire habitats previ- being harmed by temephos, showed similarity to ously treated with copepods. Macrocyclops, Mesocyclopr, and Acanthocyclops sp. that thrived in the presence of B.t.i. or permeth- rin (Riviere et al. 1987, Marten et al. 1993) or of "fhe Ae. aegypti larval production in tire habitats B.t.i. and methoprene (Tietze et al. t994). in the St. James area is shown in Fig. 2. The pres- Drums and tires are among the most cornmon ence of either species of copepod (M. longisetus or and most productive containers for Ae. aegypti in M. aspericornds) seeded with 100 or 2OO copepods Trinidad and Tobago (Rosenbaum et al. 1995). The in 2 liters of water made a signiflcant (F : 6.95, present data show that although copepods are use- df : 8, P : O.OOO1)difference for Ae. aegypti lar- ful tools against Ae. aegypti in these 2 types of val survival in tires when they were compared to containers, copepods only showed low to moderate the control (untreated tires). Means were 8.4 and perfonnance (3.8-63.IVo control) against Cx. quin- 8.5 mature larvae per tire in M. longisetus-treated quefasciatus. In this respect our results appear to tires and 16.8 and 12.0 larvae per tire in M. asper- agree with Riviere and Thirel (1981) and Kay et al. icornis-teated tires compared to 112.9 larvae per (1992), who found that Cr. quinquefosciat s were tire in the control tires. There were no signiflcant not greatly affected by Mesocyclops leukarti pilosa differences between the 2 copepod species or dos- (Daday) and M. aspericornis, respectively. Kay et age treatments (F : 2.44, dt: l, P: 0.12). There al. (1992) did find, however, that the larger M. lon- were no differences in production of Cx. quinque- gisetus was efficacious againstAe. aegypti, An. far- fasciatus based on species of copepod or initial dos- auti, aurrdCx. quinquefasciatus. age of copepod per habitat (Fig. 2). In fact, the The bacteria isolated from M. longisetus and M. control habitats were consistently lower producers aspericornis, whether maintained indoors or out- of this mosquito than the habitats treated with co- doors or from their watery medium, are generally pepods. regarded as commensals in humans. We do not Microbial studies done on M. longisetu.r main- know whether these bacteria were located internally tained indoor and outdoor at CAREC showed the or externally on the chitin of the copepods. Mem- presence of Aeromonas sobria, Enterobacter sp., bers of the Enterobacteriaceae are widely distrib- and Pseudomonas sp. Mesocyclops aspericornis uted in soil and plants as well as being normal col- homogenates grew A. sobria, Alcalignes sp., gram- onizers of the intestinal tract of humans and ani- positive bacilli, gram-positive cocci, and Pseudo- mals. The major intestinal tract pathogens such as monas pseudoalcalignes. Watery media from M. Salrnonella, Shigella, and Vibrio cholerae, whitclr longisetus culture produced gram-positive rods and has a particular affinity for chitin (Nalin et al. cocci as well as P. pseudoalcalignes artd gram-neg- 1979), were not found in our samples. However, ative rods. Aeromonas spp., which are ubiquitous inhabitants of water and soil, may cause disease in amphibians, reptiles, and fish; A. sobria has been associated DISCUSSION with extraintestinal infections in humans (Janda and The availability of mosquito-larvivorous cope- Brenden 1987). However, the risk of such infection pods in the Caribbean region offer a good promise is minimal in the utilization of water treated with for control of Ae. aegypti now that appropriate copepods. Despite this, the use of copepods forAe. strains of Macroc-vclops and Mesocyclops lrave aegypti control is currently being recommended for been identified and assayed. The 84-86Vo control non-drinking water containers only. of Ae. aegypti by M. aspericornis (Oropouche) and The present work shows that there are several M. albidus are reminiscent of the 99.9Vo control of Caribbean strains of copepods capable of being in- Ae. aegypti by M. longisetur (Marten et al. 1992). corporated alone or in association with organo- Our results are also similar to those of other work- phosphorous insecticides for Ae. aegypti control Mnncs 1997 Copepoos es ,0,BrocoNtnol Toot- ZJ and thus dengue management. These copepod control programs in the Caribbean and selected neigh- strains could be mass-produced in the various Ca- boring countries. J. Am. Mosq. Conrol Assoc. 9:1-7. 1994. Dengue and ribbean countries, and even by local cornmunities, Pan American Health Organization. dengue hemorrhagic fever in the Americas: guidelines and distributed within neighborhoods for Ae. ae- for prevention and control. Scientific Publ. 548. Wash- gypti control programs, as was suggested by Marten ington, DC. et al. (1992) in Honduras. Such use of copepods in Rawlins, S. C. and J. O. Hing Wan. 1995. Resistance in control programs could be achieved with appropri- some Caribbean populations of Aedes aegypti to several ate training and education. Vasconcelos et al. insecticides. J. Am. Mosq. Control Assoc. l1:59-65. (1992) found that in Brazil many residents, es- Rawlins. S. C.. G. G. Clark and R. Martinez. 1991. Ef- pecially school children, became very interested fects of single introduction of Toxorhynchites mocte- and participated in copepod production and use. zuma upon Aedes aegypti on a Caribbean Island. J. Am. Mosq. Control Assoc. 7:7-10. Once the trained community has a part in copepod Riviere, E and R. Thirel. 1981. La predation du copepode production and distribution, the use of the biocon- Mesocyclops leuckarti 2llosa (Crustacea) sur les larves trol tool could become a cheap, sustainable, and de Aedes aegypti et de Ae. polynesiensis: essais prelim- efficacious Ae. aegypti management system for the inaries d'utilisation comme agent de lutte biologique. Caribbean. Entomophaga 26 :427 - 439. Riviere, E, B. H. Kay, J. M. Klein and Y. Sechan. 1987. Mesocyclops aspericornis and Bacillus thuringiensis ACKNOWLEDGMENTS var- israelensis for biological contol of Aedes and Cu- /er vectors breeding in crabholes, treeholes and artificial We are grateful to our collegues at CAREC, es- containes. J. Med. Entomol. 24:425-43O. pecially A. Aasgarali and J. Ou Hing Wan, and the Rodriguez, M. L. 1992. Biological control of Aedes ae- Insect Vector Control Officers of the Port-of-Spain gyprl using copepods, pp. t45-150. /n: S. B. Halsted (eds.). Health Authority who assisted with this project and and H. 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