Predation efficiency of indigenous larvivorous fish species on Culex pipiens L. larvae (Diptera: Culicidae) in drainage ditches in Argentina

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1 12 Journal of Vector Ecology June 26 Predation efficiency of indigenous larvivorous fish species on Culex pipiens L. larvae (Diptera: Culicidae) in drainage ditches in Argentina Gerardo A. Marti, M. de las Mercedes Azpelicueta 1, María C. Tranchida, Sebastián A. Pelizza, and Juan J. García Centro de Estudios Parasitológicos y de Vectores-Cepave (UNLP-Conicet), 2 No. 584, (19) La Plata, Argentina 1 Division of Vertebrate Zoology, Museum of Natural Sciences, Paseo del Bosque, Universidad Nacional de La Plata, 19 La Plata, Argentina Received 19 September 25; Accepted 3 November 25 ABSTRACT: Two neotropical freshwater fish species, Cnesterodom decemmaculatus (Poeciliidae) and Jenynsia multidentata (Anablepidae), were collected from human-made ditches, a common habitat of the house mosquito Culex pipiens in La Plata, Argentina. Cnesterodom decemmaculatus was recorded in 62 of the 1 examined ditches, whereas J. multidentata was collected from only 21 ditches sympatrically with C. decemmaculatus. Culex pipiens was the only mosquito species collected, and its larvae and pupae were found in 38 of the 1 ditches. Fish and mosquito larvae and pupae were collected together in only two ditches and were significantly negatively correlated. Siphons of larval Culex and remnants of chironomid larvae, copepods, aquatic mites, and fish were present in the gut contents of two C. decemmaculatus from mosquito-positive ditches, while diatoms and filamentous algae were recorded in every fish dissected. Adult C. decemmaculatus and J. multidentata needed approximately 6.2 h to completely digest one Cx. pipiens 4 th instar larva under laboratory conditions. When fish were confined with a density of 6 or fewer Cx. pipiens 4 th instar larvae, C. decemmaculatus and J. multidentata adults consumed 1% of them in one day but only 35% and 42%, respectively, when confined with 15 larvae. Eradication of Cx. pipiens from a ditch, where densities had averaged 25 immatures per dip, was achieved 17 days after the introduction of 1,7 C. decemmaculatus. Journal of Vector Ecology 31 (1): Keyword Index: Culex pipiens, larvivorous fish, Cnesterodom decemmaculatus, Jenynsia multidentata, mosquito control. INTRODUCTION Culex pipiens L. is a serious nuisance and pest mosquito in many areas of Argentina. However, the broadscale application of synthetic insecticides is often associated with high levels of insecticide resistance and environmental and health concerns. Because of these concerns, biological vector control plays an increasingly important role in integrated management strategies. Some fish species have been studied as potential biocontrol agents of larval and pupal mosquitoes. Even though it is possible to find many studies on larvivorous mosquito species (Gerberich and Laird 1968, Bay 1985, Meisch 1985, Nelson and Keenan 1992, Torrente et al. 1993, Lee 2, Martinez-Ibarra et al. 22, Hurst et al. 24), to our knowledge there are no reports from Argentina. The mosquito fish Gambusia affinis (Baird and Girard) has been the most studied fish species for use as a biological control agent worldwide, but the World Health Organization (1982) discourages the introduction of exotic species because of the potential for negative ecological consequences (CDC 1973). Human-made drainage ditches in suburban areas of many cities in Argentina are among the most common larval habitats of Cx. pipiens and huge numbers of mosquitoes emerge throughout the year (Campos et al. 1993). The objectives of this study were to identify some larvivorous fish species indigenous to Argentina and to conduct laboratory and field trials to determine the predation efficacy of these fish species on Cx. pipiens larvae. MATERIALS AND METHODS Study site A survey for larvivorous fish was conducted in 1 ditches in an area of 3.6 ha (6m x 6m) located 6 km from the city of La Plata (34º 55 S, 57º 57 W), Buenos Aires province, Argentina, from November 23 to April 24. These ditches by the sides of streets are part of a continuous system connected to the El Gato stream located 3 m from the study site, which flows into the Río de La Plata. These ditches averaged 5 cm wide and 15 cm deep and drained excess rain water and, occasionally, domestic waste water that contained detergent, soap, bleach, and grease from nearby houses. By the ditches there were areas with aquatic vegetation and/or trash. Collection techniques Fish were collected with a mesh aquatic net (25 cm x 25 cm) and transported to the laboratory in a 1-liter plastic bucket containing water from the collection site aerated by a battery-operated portable air pump. Mosquito larvae and pupae were collected with a 3 ml dipper. Densities of immature mosquito stages were calculated as averages from five dips per ditch. Fish identifications were based upon the

2 Vol. 31, no. 1 Journal of Vector Ecology 13 taxonomic keys of Ringuelet et al. (1967) and Ghedotti (1998). Digestive tract content study One hundred and thirty fish from 62 ditches were fixed in 1% formaldehyde and dissected to open their digestive tracts, which were removed and washed in a Petri dish. Gut contents were separated under a stereoscope and identified under a compound light microscope. Time for complete digestion of a mosquito larva Field-collected adult males (n=1) and females (n=1) of both fish species were individually placed in glass containers (1 cm x 1 cm x 1 cm) with 5 ml of dechlorinated water. Each fish was fed one field-collected Cx. pipiens 4 th instar larva. Three fish from each species and sex were fixed in 1% formaldehyde at 15 min intervals after feeding. Fixed specimens were dissected and their digestive tracts removed. Gut contents were washed out and examined for the presence of mosquito larval remains under a light microscope. Laboratory feeding tests Feeding tests were conducted at 25±1 C and 12:12 (L:D) photoperiod. After a period of seven days of acclimation to laboratory conditions, during which the fish from ditches were kept in dechlorinated water and fed with Cx. pipiens larvae, adult fish (n=5) were individually placed in glass containers (1 cm x 1 cm x 1 cm) with 5 ml of dechlorinated water. To determine the number of larvae consumed by a single fish in 1-h, densities from 1 to 6 (increasing by 1) fieldcollected Cx. pipiens 4 th instar larvae were added to each of the five containers allocated to each density. Surviving larvae were recorded from each container after the 1-h period. This experiment was repeated five times on different dates under similar conditions. To determine the daily consumption of mosquito larvae, single adult fish (n=5) were kept in a similar manner as the previous experiment. Densities from 1 to 15 (increasing by 1) Cx. pipiens 4 th instar larvae were added to each of the five containers allocated to each density. Surviving larvae were counted daily and removed from each container. Individual fish received the same number of larvae every 24 h during a 7-day test period. Predation efficacy Larvae and pupae of Cx. pipiens were collected with a 3 ml dipper from ditches in the study area. They were counted and transported to the ditches under study in a plastic bucket with 5 liters of site water. An average of 8, larvae per ditch were released in three ditches (average 4 m long,.5 m wide,.15 m deep) into which fish were previously recorded, but their density had not been estimated. Numbers of larvae per dip (n=5) were recorded at time (immediately after larval releases), 1, 3, and 6 h post-release from both sides of the ditches. After a 2-h post-release period, 1 fish from each ditch were collected with a net, dissected, and their gut contents examined for presence of mosquito larval remains as described previously. Field release of fish in a ditch with mosquito larvae A ditch (5 m long,.5 m wide,.15 m deep) with Cx. pipiens larvae and pupae and without fish was selected for a preliminary study to determine the predatory efficacy of fish under field conditions. Fish were collected with an aquatic net in ditches from the study area, identified, counted, and transported in a 1-liter bucket with site water for release into the test ditch. Larval and pupal densities of Cx. pipiens in the ditch were determined from daily mean numbers per dip (n=5) from a week prior to introduction until four weeks after the introduction of fish. A control ditch without fish introduction located 5 m from the studied ditch was sampled for the same time period. Statistical analysis Analysis of variance (ANOVA) and comparison of means by Tukey s test (P =.5) were utilized to compare larvae consumption by fish species with different larvae densities at 1 and 24 h. RESULTS Endemic fish and mosquito species Two species of fish were identified from the examined ditches, Cnesterodom decemmaculatus (Jenyns) (Poeciliidae) (2.2 ±.6 cm long) and Jenynsia multidentata (Jenyns) (Anablepidae) (2.6 ±.3 cm long). Fish were recorded in 62 of the examined ditches (n=1), C. decemmaculatus in all 62 fish-positive ditches, and J. multidentata in 21 of these, always with C. decemmaculatus. Culex pipiens was the only mosquito species collected in these ditches, its larvae and pupae were recorded in 38% of the ditches (n=38). Larval density ranged from less than one to a maximum of 478 per dip. Fish and mosquitoes were collected in the same ditches in only two ditches, and mosquito larval density was very low in these two, less than one larva per 1 dips. A G-test for independence with Williams correction (Sokal and Rohlf 1995) indicated a significant negative association between the presence of fish and Cx. pipiens immatures (G corr. = 98.4, 1 df, P<.1). Number of larvae preyed upon Cnesterodon decemmaculatus Larval density Jenynsia multidentata Figure 1. Predation (mean ± SD) of an adult Cnesterodom decemmaculatus and Jenynsia multidentata upon different densities from 1 to 6 of 4 th instar larvae of Culex pipiens in 1 h.

3 14 Journal of Vector Ecology June 26 Number of larvae preyed upon Jenynsia multidentata Cnesterodon decemmaculatus Larval density Figure 2. Daily consumption (mean ± SD) of Culex pipiens 4 th instar larvae by an adult Cnesterodom decemmaculatus and Jenynsia multidentata at different larval densities from 1 to 15. Digestive tract contents Digestive tract contents of 13 adult fish of the two collected species, C. decemmaculatus (n=75) and J. multidentata (n=55), were examined. Diatoms and filamentous algae were recorded in the guts of every fish dissected. Siphons (n=2) of Culex larvae, exoskeletal remains of chironomid larvae (n=2), copepods (n=2), aquatic mites (n=4), and scales and bones of fish (n=2) were found in the gut contents of two C. decemmaculatus collected from the two ditches which contained both fish and mosquitoes. Digestion times Adult males and females of C. decemmaculatus (3.5 cm long) needed an average time of 6.18 h ± 1 min (SD) and 6.18 h ± 24 min (SD), respectively while adult males and females of J. multidentata (3.5 cm long) needed an average time of 6.3 h ± 18 min (SD) and 6.18 h ± 24 min (SD), respectively, to complete digestion of a Cx. pipiens 4 th instar larva. No larval sclerotized remains were observed in their digestive tracts after that time. Predation trials Adults of C. decemmaculatus (3.5 cm long) offered different densities of Cx. pipiens larvae ate an average of 6.8 ± 2.2 (SD) larvae in 1 h when density was 1 larvae and 6.8 ± 4.4 (SD) larvae when density was 6 larvae. These means were not significantly different among the six larval densities tested (P<.5). While adults of J. multidentata (3.5 cm long) ate 8.8 ± 1.6 (SD) larvae in 1 h at a density of 1 larvae and 33.8 ± 18 (SD) larvae when density was 6 larvae in the same period of time (Figure 1). Significant differences were only obtained between densities of 1 and 6 larvae (P=.1). Daily consumption of 4 th instar Cx. pipiens larvae by C. decemmaculatus and J. multidentata adults was 1% when 6 or fewer larvae were present, decreasing to 35% (n=52) and 42% (n=63), respectively, when 15 larvae were supplied (Figure 2). Differences in daily larvae consumption by a single fish were not significant when larval density was 4 and above (P>.5). Preliminary predation trials of fish upon Cx. pipiens immatures under field conditions were done in two ways: by release of mosquito larvae and pupae into three ditches with fish and by introduction of fish into a ditch with mosquitoes. Treated Control Larvae + pupae (SD) per dip Date Figure 3. Effect of the introduction of the larvivorous fish Cnesterodom decemmaculatus on mosquito density in a ditch with immature stages of Culex pipiens. Arrow: fish introduction date.

4 Vol. 31, no. 1 Journal of Vector Ecology 15 Field-collected larvae and pupae of Cx. pipiens (n=8,) were released in each ditch with C. decemmaculatus and J. multidentata. Mean larval density at time (immediately following larval releases) was 26 ± 5 (SD) larvae per dip and decreased to.2 ±.4 (SD) larvae per dip after 1 h and larvae per dip at 3 h and 6 h post-mosquito introduction. Examinations of the gut contents of 1 fish collected 2 h after larval releases from each of the three ditches under study recovered one to three larval mosquito siphons in seven of the 1 fish dissected. Densities of Cx. pipiens varied from 243 to 31 per dip during the seven days prior to fish introduction in the test ditch (Figure 3). In early December 23, 1,7 field-collected C. decemmaculatus (adults and juveniles) were introduced into the ditch. Mean mosquito densities decreased at each sampling date thereafter until 17 days later when no immatures of Cx. pipiens were collected (Figure 3). Culex pipiens density in the control ditch varied from 57 to 114 larvae and pupae per dip during the sampling dates (November 26 to December 3) (Figure 3). Weekly monitoring continued until the end of April of 24, at which time fish persisted and Cx. pipiens remained absent in the treated ditch in contrast to the control ditch where Cx. pipiens averaged 224 ± 47 (SD) larvae per dip. Subsequent monthly monitoring until October 25 demonstrated the persistence of C. decemmaculatus and absence of mosquito larvae in the treated ditch. DISCUSSION The human-made ditches studied are important habitats for the immature stages of the serious mosquito pest, Cx. pipiens, whose adults disperse into the surrounding residential areas. This mosquito species is also an important vector of West Nile virus in North America, from where this invasive arbovirus may be carried southward by migratory birds (Hayes et al. 25). Cnesterodom decemmaculatus and J. multidentata are two fish species occurring exclusively in the southern part of the Neotropical region. Although these two have been previously mentioned as larvivorous (Ringuelet et al.1967), our report provides the first data on the predatory capacity and biological control effects of these fish species. Examinations of the contents of digestive tracts indicated that the two fish species consume a broad diet. The scarcity of mosquito larval remains in guts of field-collected fish is best explained by the paucity of mosquito larvae in ditches with fish. Indeed, the highly significant negative association between fish and mosquito presence in untreated ditches indicates efficacious natural biocontrol wherever these predators can gain access to their culicid prey. Results obtained in the laboratory feeding tests confirmed that C. decemmaculatus and J. multidentata are efficient larvivores under these conditions. The different shapes of functional responses of the two predators (Figure 1) suggests different strategies in prey attack and handling. C. decemmaculatus could attack a lesser number of prey than J. multidentata in an hour. Their daily larval consumption capacities recorded in laboratory tests were comparable to results on predatory capacities of other fish species (Rees 1958, Shim and Self 1973, Menon and Rajagopalan 1977, Ikemoto et al. 1986, Fletcher et al. 1993), although the decrease in prey capture at high mosquito densities could be a laboratory artifact. Although physical and chemical characteristics of the water from these ditches were not determined in this study, the domestic effluents dumped into them suggests that these two fish species might tolerate some water pollution, which often limits the distribution of predatory fish (Weiser 1991). In addition, obstacles such as tin cans, bottles and plastic bags, and/or small patches of dense aquatic vegetation often blocked the flow of water from ditches with these fish species to adjacent ditches with mosquito larvae. Information on the pollution tolerance, biology, and ecology of these two larvivorous fish is scarce and more research is necessary in order to use them efficiently in future programs of integrated control in larval habitats of Cx. pipiens. However, this study demonstrates that transferring fish among ditch habitats or clearing obstacles to water flow could augment the biological control of Cx. pipiens by these two fish native to Argentina. Acknowledgments We are grateful to Phillip Lounibos for critical and constructive comments on an early version of the manuscript. This research received financial support from the Comisión de Investigaciones Científicas de la Provincia de Buenos Aires-CIC, University of La Plata and Conicet, Argentina. 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