Environ Biol Fish (2012) 94:639–647 DOI 10.1007/s10641-011-9970-4

Some ambient environmental conditions, food and reproductive habits of the banded lampeye killifish Aplocheilichthys spilauchen in the Kakum estuary wetland, Ghana Isaac Okyere

Received: 17 May 2011 / Accepted: 22 November 2011 / Published online: 10 December 2011 # Springer Science+Business Media B.V. 2011

Abstract The use of killifish in biological control of mosquitoes to complement other mosquito control programs is gaining interest due to the increasing development of chemically resistant substrains of mosquitoes. This study investigates the ambient salinity, temperature and dissolved oxygen as well as food habits, sex ratio, fecundity and spawning frequency of Aplocheilichthys spilauchen in Ghana in an effort to broaden knowledge of the suitability of this killifish for mosquito control. Sampling was undertaken from July 2009 to January 2010 to monitor the levels and variations in the environmental parameters and fish abundance, and also to determine the feeding ecology, fecundity and possible spawning frequency. Results suggested that lower levels of salinity favoured the population while levels beyond 4‰ were detrimental. The species preyed highly on insects and insect larvae, which constituted 80% of food consumed. Females significantly outnumbered males by almost 2:1 (χ2 =28.57, P<0.05), and had low fecundity (2– 44 eggs). Ova diameter analysis suggested that the species spawns continuously. The better survivability of the species in freshwater pools with continuous spawning of drought tolerant eggs, and the high preference for insects and insect larvae suggest that it could be a useful candidate for biocontrol of mosquitoes. I. Okyere (*) Department of Fisheries and Aquatic Sciences, School of Biological Sciences, University of Cape Coast, Cape Coast, Ghana, West Africa e-mail: [email protected]

Keywords Killifish . Environmental requirements . Feeding habits . Breeding habits . Mosquito control

Introduction The use of pesticides for mosquito control has been an important component of mosquito control program around the world. However, the increasing development of chemically resistant substrains of mosquitoes, coupled with the ecosystem damage caused by chemicals used in the vector control process, has renewed interest in biological control alternatives to complement other mosquitoes control programs (Dabire et al. 2008; Munhenga et al. 2008). According to Hamouda and Samraoui (2007) as well as Matias and Adrias (2010), the mosquito fish (Gambusia affinis) was eventually selected for worldwide mosquito control because of its high larvivorous capacity, high fecundity and adaptability to new environments, but was not successful due to its intrinsic aggressive nature which nearly resulted in the extinction of many other native species. Consequently, the focus of recent efforts point to developing indigenous and less aggressive aquarium fishes as alternative in providing mosquito larval control in streams and pools (Ghosh et al. 2004; Howard et al. 2007). Killifishes (a group of small Cyprinodontiform fishes), native to South America and Africa (Ghedotti 2000), have recently gained much attention as suitable candidates in the pursuit of mosquito biocontrol especially in

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ephemeral aquatic environments such as seasonal pools. Their suitability lies in the fact that most species feed on insect larvae and small invertebrates from water surface (Holden and Reed 1991; Dankwa et al. 1999), and are non-aggressive when mixed with other species (Matias and Adrias 2010). Most importantly, they have the ability to survive prolonged drought by producing diapausal embryos which are highly resistant to environmental extremes such as drought, high temperature, anoxia and physical as well as chemical damage (Matias 1982; Holden and Reed 1991; Podrabsky et al. 2001). When seasonal pools dry, the diapausing embryos survive in the dry substrate, and hatch at the start of the rains to re-populate the pools. As indicated by Matias and Adrias (2010), this life cycle which coincides with that of their potential prey such as mosquitoes that also begin their life cycle in the same seasonal pools provides a compelling reason for the interest in the potential of killifish for vector control in seasonal habitats. Regardless of this growing interest, few studies have looked at the prospects of some killifish for this purpose in Africa (Ghosh et al. 2004; Howard et al. 2007), with the well explored ones being species of the genus Nothobranchius common in Tanzania (Matias and Adrias 2010). However, there are other species that have a wider geographical distribution on the continent that may also be suitable candidates in the localities where they occur, but have remained unexplored. The banded lampeye Aplocheilichthys spilauchen (Poeciliidae) is one of the killifishes in Africa that have not been explored for their mosquito control potential, despite the prevalence of the fish in coastal swamps, river mouths, lagoons and mangrove swamps from Senegal to Angola (Wildekamp 1995). Reports have indicated that survivability under freshwater conditions, larvivorous feeding ecology and some reproductive habits including fecundity and spawning frequency are among the major determinants of the suitability of a killifish for the biocontrol course (Podrabsky et al. 2001; Hamouda and Samraoui 2007; Matias and Adrias 2010). Therefore, the consideration of A. spilauchen as a probable candidate pertinently requires knowledge of its environmental requirements especially salinity tolerance, as well as its food and breeding habits.

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However, the few works available on the species tend to concentrate on the taxonomy (Parenti 1981; Ghedotti 2000) and the localities of distribution (Wildekamp et al. 1986; Wildekamp 1995; Huber 1996) with marginal reference to their environmental requirements, feeding ecology and reproduction. This paucity of information largely constrains knowledge of the potential of the fish as a control agent. Against this background, the present study investigates the ambient salinity, temperature and dissolved oxygen as well as food habits, sex ratio, fecundity and spawning frequency of a wild population of A. spilauchen in Ghana (West Africa) as a contributory effort in determining the ecological and biological suitability of the species in malaria vector control in Africa.

Materials and methods Study area The Kakum estuary wetland is a coastal marsh located about 2 km west of Cape Coast in the Central Region of Ghana (approximately 5o 6′ N; 1o 18′ W) and is associated with the Kakum River Estuary which lies to the west of the wetland (Fig. 1). Three main grasses dominate the area: the saltwater couch Paspalum vaginatum (Poaceae), the sedge-grass Cyperus articulatus (Cyperaceae) and the bulrush Typha australis (Typhaceae). In the rainy or wet season (from May to September) flood waters from the Kakum River inundate the wetland and fish become abundant while the area is characterized by isolated natural pools during the dry season (October to late March). Measurement of environmental parameters and sampling of the fish species Measurement of environmental factors and collection of fish samples were undertaken monthly in five relatively large pools (surface area range from 661 m2 to 2680 m2; depth range from 0.6 m to 1 m) from July 2009 to January 2010 between the hours of 10:00 and 13:00 GMT. Salinity, temperature and dissolved oxygen were recorded with a Water quality checker (Model: Horiba U-10 meter) by immersing the probe

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Fig. 1 Map of Ghana showing the Kakum Estuary Wetland

into the pool. Three measurements of each parameter were taken at different points in each pool on a sampling date and the average value calculated. A pole-seine net (7 m long and 1.5 m deep) with stretched mesh size of 5 mm was used for the fish sampling; one seine per pool per month. The specimens were preserved in 10% formalin soon after capture to arrest post mortem digestion of stomach contents and transported to the laboratory for further examination. Identification was undertaken using Paugy et al. (2003) and the number of individuals collected from each pool was recorded. The total length (TL) (measured to the nearest 0.1 cm) and body weight (BW) (recorded to the nearest 0.1 g) of each specimen was determined and the specimens were dissected to determine the sex after which the stomach content was examined using dissecting and compound microscopes. Ripe ovaries were preserved in 10% formalin for studies on the fecundity and ova diameter measurements. Eggs were teased from the ovarian tissue in a petri dish and the immature ova were separated from mature ones prior to counting the latter. The whole count method (Bagenal and Braum 1978) was employed in determining the absolute fecundity of fish.

Analysis of data The statistical significance of the difference in environmental factors among the months was evaluated for each of the three parameters using the F-test or Analysis of Variance (ANOVA) (Zar 1999). Size distribution of the population was analysed at 0.5 cm class intervals to establish their modal sizes. Stomach contents were analysed using the “points” and the frequency of occurrence methods (Hyslop 1980; Lima-Junior and Goitein 2001) on a four point scale; 10 points for full stomach, 7.5 for three-quarters, 5 for half and 2.5 for quarter filled stomach. The percentage composition of each of the food items was estimated. Sex ratio of the species was calculated as the ratio of the number of males to females, and a Chi-squared test (Zar 1999) was used to verify the significance of numerical differences between the sexes. The relationships between fecundity and total length; and fecundity and body weight were established using a regression analysis and the statistical significance of the relationships was verified with the Student’s t-test (Zar 1999). The diameter of eggs of 5 female specimens was measured with stage micrometer to the nearest 0.1 mm

642 Table 1 Monthly changes in mean salinity temperature and dissolved oxygen (standard error in parenthesis) and A. spilauchen abundance in the Kakum Estuary wetland (means were estimated with fifteen replicates from the five pools and represent a sampling date in the month)

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Month

Jul-2009

Salinity (‰)

Temperature (°C)

Dissolved oxygen (mg/l)

No. of A. spilauchen specimens encountered

0.7 (±0.2)

28.0 (±1.1)

5.0 (±0.3)

287

Aug-2009

1.6 (±0.1)

30.6 (±0.7)

4.9 (±0.4)

235

Sept-2009

2.0 (±0.1)

34.3 (±0.9)

4.9 (±0.2)

178

Oct-2009

3.4 (±0.2)

40.2 (±0.6)

5.2 (±0.3)

54

Nov-2009

3.9 (±0.1)

41.3 (±0.5)

5.4 (±0.6)

2

Dec-2009

4.7 (±0.1)

43.6 (±0.7)

5.4 (±0.5)

1

Jan-2010

5.9 (±0.2)

46.6 (±0.5)

5.7 (±0.6)

0

to determine the possible frequency of spawning of the species (James and Baragi 1980).

Results Variations in environmental conditions and fish abundance A total of 757 specimens were collected during the survey. Data from the five pools were combined for analysis after it had been ascertained that there was no appreciable difference among the pool samples in both environmental and fish data. As shown in Table 1, salinity and temperature increased progressively from the wet season to the dry season, with monthly increments being significant (F=5.6, P>0.05 for salinity; and F=5.1, P>0.05 for temperature). On Fig. 2 Length-frequency distribution of A. spilauchen in the Kakum Estuary wetland

the other hand, A. spilauchen abundance declined continuously during the period, with their extermination from the pools coinciding with temperatures around 41.3°C and salinities around 3.9‰ (November 2009) during which fish mortalities were observed and some birds were seen preying on the dead fish. Dissolved oxygen however remained fairly uniform around an average of 5 mg/l throughout the study and did not differ significantly among the months of the study (F=0.51; P>0.05). Size distribution Male specimens ranged from 3.0 cm to 5.6 cm TL while females measured 2.4 cm to 5.4 cm TL suggesting that the males were slightly bigger than the females. As illustrated in Fig. 2, the population was dominated by individuals of the 3.0–3.4 and 3.5-3.9 cm classes.

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Fig. 3 Frequency and composition of different food items in the diet of A. spilauchen from five pools in the Kakum Estuary wetland

Food habits of the population A total of 757 specimens were examined; 133, 136, 92, 176 and 220 from Pools I–V respectively of which 3 from Pool II and 6 from Pool V had empty stomachs. Debris, and larvae and adult of insects of the order Diptera were the main food of the fish (Fig. 3). The most consumed food of the population was larvae of an unidentified dipteran which was ingested by 60–80% of the individuals in the pools, and constituted 20–40% of the bulk food consumed. An adult dipteran insect which could also not be identified

were eaten by 60–98% of the specimens and constituted between 30% and 40% of the eaten prey while approximately 65–80% of the fish took debris which made up a considerable proportion of their diet. Chironomid larvae (Diptera) and copepods made up a very small contribution to the bulk food. Sex ratio and fecundity Of the 757 specimens examined, 244 were males while 442 were females, with the remaining 71 being juveniles whose sex could not be determined. The

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0.96 mm–1.05 mm and the third by eggs measuring 1.35 mm–1.45 mm.

Discussion

Fig. 4 Regression plot of fecundity-total length relation of A. spilauchen population in the Kakum Estuary wetland

females therefore significantly outnumbered the males in a ratio of 1:1.81 Male: Female (χ2 =28.57, P<0.05). The gravid females encountered in the samples measured 3.6 cm to 5.4 cm and had an absolute fecundity range of 2 to 44 eggs per female. As shown in Figs. 4 and 5, fecundity weakly correlated with total length (r= 0.45) and body weight (r=0.48) in linear relationships, and the correlations were not statistically significant (t=1.4, P>0.05 for length; and t=1.2, P>0.05 for weight).

Ova diameter frequency Three modes or peaks were consistently observed in the ova diameter distribution of each of the five specimens (Fig. 6), with the first being constituted by ova of diameter 0.65 mm, the second by ova measuring

Fig. 5 Regression plot of fecundity-body weight relation of A. spilauchen population in the Kakum Estuary wetland

It has been explained by Welcomme (1975) that sustained increase in water temperatures enhances evaporation in coastal floodplain pools which consequently engenders increased salinities. This possibly explains the parallel rise in both parameters during the study. Aplocheilichthys spilauchen is reported to tolerate temperatures within a range of 24°C to 32°C (Riehl and Baensch 1991) which clearly falls below the 40°C recorded in October 2009 and beyond. However, the higher temperatures would not significantly hinder their re-population cycle during the mosquito control process since the diapausal embryos are resistant to high temperatures (Matias 1982; Podrabsky et al. 2001). Although reports indicate that A. spilauchen inhabits both fresh-and-brackishwater habitats (Huber 1996; Paugy et al. 2003), results of this study suggest the species as being more of a freshwater than euryhaline; given the incessant decline in fish numbers with increasing salinity during the study. It is worthy to note that the mortalities were observed not only for the banded lampeye but also other freshwater fishes while the brackishwater species such as Sarotherodon melanotheron and Liza falcipinnis survived and remained abundant during the period (See Okyere 2010). Conceivably, the coincidence of salinities beyond 4‰ with lampeye mortalities in November 2009 is an indication that lower salinities are favourable for their survival while levels beyond 4‰ are intolerable to the fish. This could explain why Adite (2002) encountered high numbers of the species in the riverine portions of the Mono River in Benin, and none in the estuarine areas. Since survivability in freshwater is a major determinant of the suitability of a killifish for mosquito control (Hamouda and Samraoui 2007), the banded lampeye could be a probable species for this control measure. Although killifishes are generally known to feed on small aquatic invertebrates (Schultz 2004), high preference for mosquitoe larvae is one of the ideal feeding characteristics of Nothobranchius guentheri identified as good candidates for biocontrol of mosquito breeding (Matias and Adrias 2010). The A.

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Fig. 6 Ova diameter frequency distribution of A. spilauchen from the Kakum Estuary wetland

spilauchen population preyed highly on insects and insect larvae (chironomid and other dipteran larvae), suggesting that the trophic ecology of this species is similar to that of the other biocontrol killifish. What is unclear is whether the banded lampeye would prefer preying on mosquito larvae to others and this could be verified in experimental trials as done for N. guentheri. In fish species, egg production has been found to relate to the degree of parental care and survival rates. Fecundity is high in species that provide little or no parental care for their eggs and larvae, while those that provide a moderate or high degree of care often have low fecundity (Fuiman 2002). The low absolute fecundity of A. spilauchen could therefore be attributed to their production of drought tolerant diapausal eggs which ensures high survival of their embryos in the

ephemeral pools (Holden and Reed 1991; Dankwa et al. 1999; Podrabsky et al. 2001). This enables easy re-population in the seasonal pools at the onset of the rains; a habit of killifishes suitable for mosquito control (Matias and Adrias 2010). The low fecundity could also account for the higher number of females than males as only a few males would be of relevance in the population to produce spermatozoa to fertilize the few eggs. The weak positive correlation between fecundity and total length as well as fecundity and body weight implies that number of eggs produced is not clearly related to body size. Furthermore, the three successive modes observed in the ova diameter frequency distribution likely implies that the species spawns continuously since according to James and Baragi (1980), the occurrence of egg batches of successive

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modes in an ovary is an indication of continuous spawning habit. Such continuous breeding is pertinent for the sustenance of the population to effectively check the fast rate of mosquito breeding. Monthly length–frequency distribution is however needed to further provide insight into the spawning frequency and other recruitment habits of the species. In conclusion, the study has demonstrated that Aplocheilichthys spilauchen survived better under conditions near freshwater than brackish, with their tolerance level for salinity being lower than 4‰. The better survivability of the species in freshwater pools, the seemingly continuous spawning habit coupled with production of drought tolerant eggs which survive in ephemeral pools, and the high preference for preying on insect and insect larvae suggest that the species could be potentially useful for mosquito control. However, further semi-field and laboratory studies on their preference for mosquito larvae and details of the physiology and ontogeny of their diapausal embryos are needed to fully ascertain their suitability for the biocontrol. Acknowledgements The author is grateful to the Department of Fisheries and Aquatic Sciences of the School of Biological Sciences, University of Cape Coast, Ghana for providing vehicle, field and laboratory equipment as well as laboratory space for this research. I am also thankful to Prof John Blay and Dr. Joseph Aggrey-Fynn for their assistance in the laboratory. I finally thank the chief technician Mr. Peter Aubyn as well as the field assistants Mr. John Eshun and Mr. Benjamin Owusu of School of Biological Sciences, UCC for their tireless efforts throughout the data collection.

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