Background

Culex quinquefasciatus Say, 1823 mosquito is the common southern house mosquito. It is the principal vector of Wuchereria bancrofti, responsible for lymphatic filariasis, a debilitating public health disease (Elkanah et al., 2017; World Health Organization, WHO, 2017). The physico-chemical properties of the mosquito larval habitats determine its quality, in terms of attractiveness to gravid female mosquitoes for oviposition (Braks, Leal, & Cardé, 2007; Eneanya et al., 2018), developmental success of immature mosquitoes (Oyewole et al., 2009), species distribution (Minakawa, Mutero, Githure, Beier, & Yan, 1999) and adult life traits (Mwangangi et al., 2007). Among these physico-chemical components are water pH and hardness levels.

These components are affected by various factors such as the nature of underlying rock, agricultural inputs and anthropogenic activities (Rim-Rukeh, 2013; Robert, Awono-Ambene, & Thiolouse, 1998; Salihu et al., 2017; Singare, Lokhande, & Pathak, 2010). Having quantitative data on the influence of these two parameters on the development and fitness of mosquito vectors is important. The information will update, firstly, the current literature on biology of the vector and, secondly, the role the parameters play in biological fitness for disease transmission and, thirdly, provide information on the potential use of such parameters in vector control via environmental manipulation.

For aquatic insects, ambient pH levels influence balance in body fluid ionic composition, which is chief in maintaining homeostasis and development (Daily, Hitt, Smith, & Snyder, 2012; Gillott, 2005; Patrick & Bradley, 2000). The pH of an aquatic habitat is also a major physical factor that limits survivorship and, hence, distribution of mosquito species (Yee, Kneitel, & Juliano, 2010), affecting availability of essential mineral and food elements for the development of mosquitoes (Clements, 2000). Studies have shown that mosquito larvae are capable of adapting to and tolerating fluctuations in ionic levels in these habitats (Nadia, Thamer, & Saad, 2005; Patrick & Bradley, 2000; Wigglesworth, 1933). Even though different ranges of pH values have been reported to support development and survivorship of mosquitoes in larval habitats in the wild, there is dearth of information on systematic studies on the influence of these ranges of pH on life history traits and/or contribution to biological fitness of mosquitoes.

Water hardness, on the other hand, is produced by the presence of cations of manganese, magnesium, calcium and ferrous iron. The relative quantities of these ions in water determine its level of hardness (Regional Aquatics Monitoring Program, RAMP, 2015). These ions, depending on concentration, play physiologic roles in structural formation, protection, metabolism, developmental and survivorship in aquatic organisms (Blanksma et al., 2009; Leschen & Cutler, 1994; Molokwu & Okpokwasili, 2004; Pelizza, Lopez-Lastra, Becnel, Bisaro, & Garcia, 2007; Poteat & Buchwalter, 2014). In nature, water hardness levels determine the quality of most aquatic habitat (Milad, Mohammed, & Seyed, 2011) and productivity of mosquito habitats, especially, domestic wells (Robert et al., 1998).

Studies have shown that despite the importance of physico-chemical factors in the development of aquatic organisms, especially mosquitoes, there are permissible levels for optimal immature development, which in turn determine the quality of adult life traits. These permissibility levels are usually species- and life stage-specific; outside these limits, stress-like conditions set in, affecting growth negatively by eliciting mortality (Mgbemena & Ebe, 2012; Mpho, Callaghan, & Holloway, 2002; Ukubuiwe et al., 2018a; Ukubuiwe, Olayemi, Omalu, Arimoro, & Ukubuiwe, 2018b). Further, environmental stress conditions greatly affect feeding rates in aquatic organisms (Merritt, Dadd, & Walker, 1992; Timmermann & Briegel, 1993), with its attendant reduction in metabolic energy and reserves (Timmermann & Briegel, 1999).

Unfortunately, in mosquito bioecology, data gathered from field observations on water pH and hardness levels usually form the basis for extrapolating their effects on bionomics of mosquitoes (Garba & Olayemi, 2015; Mwangangi et al., 2007). These data, though important, are not sufficient in understanding the actual contributions of these critical larval breeding factors to mosquito growth, hence the need for this study.

In the present study, a systematic report on the influence of varying levels of water pH and hardness levels (separately) on developmental successes and adult fitness attributes in Cx. quinquefasciatus mosquito was investigated. This is to provide first-hand quantitative information on the contribution of these critical micro-environmental larval habitat factors to vectorial success of mosquito species.

Materials and methods

Preparation of water media

Seven water pH levels and five hardness levels were prepared following standard procedures. The pH values (4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) were prepared according to techniques of Pelizza et al. (2007) and Ivoke, Mgbenka, and Okeke (2007), with slight modification. The levels were measured using a digital pH meter (model: Jenway 3305).

The water hardness regimens (0 (soft), 30 (slightly hard water), 90 (moderately hard water), 150 (hard water) and 210 mg/L CaCO3 (very hard water)) were prepared based on standard classification of water hardness as described by Milad et al. (2011) and reported by Ukubuiwe et al. (2018c).

Source, rearing and maintenance of experimental insects

Culex quinquefasciatus mosquitoes were collected from a colony of the species maintained in the Insectary Unit of the Department of Animal Biology, Federal University of Technology, Minna. First larval instars of the mosquito species were reared in the prepared artificial (pH and hardness) water media according to the methods of Ukubuiwe et al. (2012). The pupae on pupating were placed in plastic bowls half-filled with the artificial media and kept in adult-holding cages until emergence. The adults were maintained on 10% sucrose solution (Ukubuiwe, Olayemi, Omalu, Arimoro, Baba, et al., 2018).

Entomological parameters measured

The effects of water pH and hardness levels on the immature survivorship, duration of development and larval growth rates of the mosquitoes were recorded daily at a 12-h interval (Timmermann & Briegel, 1999; Ukubuiwe, Olayemi, & Jibrin, 2016). After emergence, the numbers of emergent male and female mosquitoes were counted (while fed 10% sucrose solution). The longevity and daily mortality of the adult mosquitoes were recorded (Ukubuiwe, Olayemi, Omalu, Arimoro, Baba, et al., 2018). Adult fitness attributes of the mosquitoes were estimated from the wing lengths (Ukubuiwe, Olayemi, Omalu, Jibrin, & Oyibo-Usman, 2013).

Data analysis

Data generated from the independent studies were processed into means and standard deviation (mean ± SD) using Microsoft Office Excel 2016 and Statistical Package for Social Scientists (SPSS) version 21. Differences between means of any two entomologic variables (e.g. between the wing lengths of male and female mosquitoes from regimens of a physico-chemical factor) were compared using Student t test, while those among entomologic variables (e.g. among sexes from the various regimens of a physico-chemical factor) were compared for significant difference using one- or two-way analysis of variance (ANOVA), as appropriate. The differences in means were separated using Duncan multiple range test (DMRT). All decisions on the level of significance for statistical comparison of means were assumed at p = 0.05.

Results

Effects of rearing-water pH and hardness levels on the duration of development and larval growth of Culex quinquefasciatus

Analyses revealed that extreme pH and hardness conditions had significant negative effects on developmental times and larval growth rates of the species (Table 1 and Fig. 1a, b). Larvae reared at pH 7.0 had the shortest duration of development (i.e. fastest development), while those at extreme acidic (4.0) or alkaline (10.0) pH conditions had, significantly (df = 6, p < 0.05), increased duration of development (Fig. 1a). As water hardness level increased, the duration of the development of the species gradually increases, with cohorts reared at the highest concentration (210 mg/L CaCO3 spending the longest time as immature (Fig. 1b). Unlike the larvae, developmental times of pupae were not affected at all pH (df = 6, p = 0.204) and hardness (df = 4, p = 0.181) conditions tested (Table 1).

Table 1 Effects of water pH and hardness levels on the duration of the development and growth rate of Culex quinquefasciatus
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Fig. 1
figure 1

Bars followed with the same letter are not significantly different at p < 0.05 according to Duncan multiple range test following ANOVA and represent mean (± SD). a Effects of water pH on developmental times of larval instars (I–IV) of Culex quinquefasciatus.b Effects of water hardness on developmental times of larval instars (I–IV) of Culex quinquefasciatus

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Developmental time (i.e. total larval + pupal durations) also followed similar pattern as the larval life stages, i.e. delayed development at extreme water conditions. At pH 10.0 and 210 mg/L CaCO3, the species had the longest duration of development. On the other hand, immature mosquitoes reared at pH 7.0 and 0 mg/L CaCO3 had the shortest developmental times (Table 1).

Larval growth rate (LGR) of the species was also significantly affected by both water conditions (i.e. pH and hardness). The larval size increased as pH tilted towards neutrality. However, with increasing alkaline conditions, larval size started reducing and was lowest at pH 10.0 (Table 2). Similarly, water hardness condition had an inverse relationship with LGR, i.e. increase in hardness resulted in reduced LGR, although at lower hardness levels (≤ 30 mg/L CaCO3) analyses revealed no significant difference (df = 4, p = 8.14) in LGR.

Table 2 Effects of water pH and hardness levels on growth rate of Culex quinquefasciatus
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Effects of rearing-water pH and water hardness level on life-stage survivorship of Culex quinquefasciatus

Analyses revealed significant effects of increasing pH and hardness conditions on survivorship of all life stages of the species (Fig. 2a, b and Table 3). Larval instar survivorship was the highest at pH levels of 5.0–8.0, and the lowest at pH 4.0 and 10.0 (Fig. 2a). Similarly, hardness levels ranging from 0 to 90 mg/L CaCO3 (i.e. ≤ moderately hard water) supported the highest survivorship (Fig. 2b). Among larval instars, LI was the most affected, especially at the highest concentration tested (i.e. 210 mg/L CaCO3). Pupal survivorship, on the other hand, was the highest at pH 8.0 and the lowest at pH 10.0 (Table 3).

Fig. 2
figure 2

Bars followed with the same letter are not significantly different at p < 0.05 according to Duncan multiple range test following ANOVA and represent mean (± SD). a Effects of water pH on daily survivorship of larval instars of Culex quinquefasciatus.b Effects of water hardness on daily survivorship of larval instars of Culex quinquefasciatus

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Table 3 Effects of water pH and hardness level on life stages’ survivorship of Culex quinquefasciatus
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Daily adult survivorship of the species reduced significantly with the increase in physico-chemical conditions. At extreme acidic (pH 4.0) and alkaline (pH 10.0) conditions, survivorship was the lowest and was respectively 51.93 ± 16.30 and 61.13 ± 7.24%; meanwhile, at pH range of 5.0–9.0 (range = 76.16 ± 2.29–82.43 ± 1.52%), adult survivorship was the highest (Table 3). Likewise, adult mosquitoes reared at 0 to 90 mg/CaCO3 (i.e. ≤ moderately hard water) had the highest survivorship (range = 74.48 ± 6.20 to 78.16 ± 7.34%), while those reared in ‘very hard’ water condition (210 mg/L CaCO3) had the lowest (44.93 ± 8.41%) (Table 3).

Generally, survivorship among sexes decreased with the increase in water conditions (Fig. 3). However, between sexes in a factor, male (♂) mosquitoes were more affected than their female (♀) counterparts. In water pH treatments, ♂ mosquitoes (range = 33.99 ± 39.39 to 80.92 ± 3.50%) were more affected than the ♀ mosquitoes (range = 57.86 ± 13.08 to 84.35 ± 1.70%). Similar trends were seen among water hardness treatments. The range for ♂ mosquitoes is 69.78 ± 7.20 to 73.94 ± 4.08% and for ♀ mosquitoes, 51.80 ± 22.74 to 80.72 ± 8.01%.

Fig. 3
figure 3

Biplots of effects of water pH and hardness on daily survivorship of male and female adult life stages of Culex quinquefasciatus

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Effects of rearing-water pH and hardness on emergence success and longevity of Culex quinquefasciatus

Analyses showed significant (p < 0.05) negative effects of extremes of pH on emergence success and longevity of the species, as the least numbers of adult emerged at pH 4.0 and 10.0. On the other hand, the highest emergence success rates were obtained from pH range of 5.0 to 9.0 (range = 46.75 ± 2.05 to 49.63 ± 0.52 adults/50 larvae) (Table 4). Water hardness showed similar inverse relationship with these parameters, with increased water hardness conditions, significantly, reducing emergence success (range = 10.38 ± 3.16 to 88.00 ± 4.11 adults/100 larvae) (Table 4).

Table 4 Effects of water pH and hardness levels on emergence rates and longevity of adult life stage of Culex quinquefasciatus
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Post-emergence longevity (PEL) of the adult mosquitoes was also affected by pH regimens. Adult mosquitoes from water media with pH range of 6.0–8.0 lived the longest (> 18 days), whereas pH conditions below or above this range (i.e. < pH 6.0 or > pH 8.0) reduced adult longevity. The shortest longevity was observed in mosquito cohorts from pH 4.0 and 10.0 (Table 3). Mosquito cohorts from hardness regimen of 0–90 mg/L CaCO3 lived longer (range = 15.07 ± 1.17 to 16.16 ± 0.80 days) than those from higher hardness levels (< 10 days) (Table 4).

Discussion

Effects of rearing-water pH and hardness conditions on duration of development and larval growth rate of Culex quinquefasciatus

In the present study, rearing-water with pH values ranging from 4.0 to 10.0 was used to rear immature Cx. quinquefasciatus mosquitoes to determine the effects on the duration of the development and larval growth rate of the species. The result showed a growth regulating effect of pH on the species, with extreme pH values eliciting, significantly (df = 6, p < 0.05), longer durations of development of the species. Those reared at pH 7.0 were the fastest developer, while those reared at pH values of 4.0, 9.0 and 10.0 were the slowest. Earlier, Woodhill (1942) and Tanimura and Sonoji (1952) reported that pH ranges of 3.6–4.2 increased the development times in Aedes aegypti).

This ability of Cx. quinquefasciatus to develop in the pH ranges tested, though with decreased survivorship at extreme pH conditions, may suggest possible tolerance of the species to a wide range of pH conditions. Although there is little or no field information to support the development of the species at all the water pH ranges studied, field reports from the study area (Minna) and areas with similar ecotype suggest that pH range of 6.0 to 8.0 is ideal for the development of the species in nature (Garba & Olayemi, 2015; Ikeh et al., 2017; Olayemi, Omalu, Famotele, Shegna, & Idris, 2010; Salihu et al., 2017). This study, thus, peeks into resultant effects of these pH ranges on the species (Cx. quinquefasciatus) in conditions, where the habitats have been altered, passively, by anthropogenic activities or, actively, as a vector control strategy.

Species-specific range of pH conditions for the development of other mosquito species has been reported and included 3.3 to 8.1 for Ochlerotatus taeniorhynchus, 4.4 to 9.3 for Aedes geniculatus and Anopheles plumbeus, 3.3 to 9.2 for Psorophora confinnis, 2 to 9 for Aedes flavopictus and 2 to 10 for Armigeres subalbatus (Christophers, 1960). In the present study, the duration of the development of all immature life instars and stages (i.e. larval instars and pupae) of Cx. quinquefasciatus was significantly affected by water hardness. As water hardness level increased progressively, from 0 to 210 mg/L CaCO3, there was a significant delay in the development. The species spent about twice the developmental time at 0 mg/L CaCO3 to complete the development at 210 mg/L CaCO3. This shows that the quantity of CaCO3 present in a mosquito larval breeding habitat, though necessary for normal development, can determine the time taken to reach adulthood.

In this study, moderately ‘hard water’ supported the fastest development of the species. Similar water conditions have been reported for optimum development in laboratory studies for other aquatic organisms (Abernathy, 2004; Blanksma et al., 2009; Milad et al., 2011; Molokwu & Okpokwasili, 2004), and in field studies for mosquitoes (Kant, Pandey, & Sharma, 1996; Mgbemena, Opara, Osuala, & Iwuala, 2009; Mwangangi et al., 2007; Olayemi et al., 2010; Piyaratne, Amerasinghe, Amerasinghe, & Konradsen, 2005).

In the present study, larval growth rate, LGR, an indicator of daily weight accumulation, was significantly affected by pH and hardness levels. For example, LGR was the highest at pH value of 7.0 and the lowest at pH 10.0. The low LGR at pH 10 may indicate impaired feeding and/or growth processes during the phagoperiods. This impairment could probably be stress-related and/or disruption of physiological process, which affects the feeding process and accumulation of tenerals (Lazareviæ, Periæ-Mataruga, Vlahoviæ, Mrdakoviæ, & Cvetanoviæ, 2004). More so, neutral pH has been reported as ideal (in nature) for breeding of Culex mosquitoes (Sattler et al., 2005).

Effects of rearing-water pH and hardness levels on life-stage survivorship of life stages of Culex quinquefasciatus

One of the most important factors that serve as an index for pollution and survivorship of an organism is pH (Singare et al., 2010). In the present study, pH values ranging from 4.0 to 10.0 supported the survivorship of Cx. quinquefasciatus mosquitoes though affecting the life stages in various ways. This effect was, however, pH level- and life-stage-dependent. Though there are no field investigations or reports suggesting that the species thrive in this range of pH values (i.e. 4.0 to 10.0), generally, in this study, extreme values of pH (i.e. either too acidic or basic) significantly reduced survivorship, with the species exhibiting some degree of adaptation/tolerance to the extreme conditions by surviving till adulthood.

Further, even though the mosquito species showed some element of survivorship in all pH regimen investigated, it had higher survivorship at pH values ranging from 5 to 9; outside these, survivorship significantly reduced. These ranges may, perhaps, indicate the survival pH range for the species. Buchman (1931) had earlier reported that larvae of Culex pipiens lived in water at pH from 4.4 to 8.5, but died beyond these values.

Although little or no reports have shown that Cx. mosquitoes thrive in habitats with pH values of 4.0 and 10.0, the present study reveals that survivorship at these values may be low. It should be noted that in controlled experimentations (laboratory conditions), mosquito survivorship is usually high, when compared with field population. However, despite these expectations, survivorship at pH 4 and 10 was still low. It, therefore, means that, perhaps, where these pH conditions occur (either naturally or induced artificially as a vector control strategy) may elicit greater mortality in the wild (i.e. field/uncontrolled condition).

The survivorship recorded for this species (in this study) at extreme pH conditions could be attributed to tolerance, acclimation and regulatory abilities of the species to these extreme conditions. This is important as several coping capabilities which play vital role in species distribution under field conditions have been reported (Bayly, 1972; Bradley, 1994; Wigglesworth, 1933) Similar abilities have been reported for two members of the mosquito tribe Aedini, Aedes aegypti and Ochlerotatus taeniorhynchus (Clark, Flis, & Remold, 2004), and Anopheles plumbeus (Christophers, 1960), with remarkably different regulatory mechanisms for dealing with ionic and stressful environment (Patrick & Bradley, 2000).

In the present study, there was a significant reduction in larval and pupal survivorship at very high hardness level (above 90 mg/L CaCO3). However, survivorship of both immature life stages was not affected by hardness level at and below 90 mg/L CaCO3 (i.e. moderately ‘hard water’).

Despite the relatively high adult daily survivorship (> 50%) recorded for the species at the pH levels tested, extreme pH conditions (i.e. pH 4.0 and 10.0) elicited lower adult survivorship. Meanwhile, pH range of 5.0–9.0 supported very high survivorship. Furthermore, survivorship between the male and female of a regimen was not significantly affected. Water hardness also significantly affected daily survivorship of adult mosquito of the species, as hardness conditions above 90 mg/L CaCO3 (i.e. moderately ‘hard water’) significantly lowered survivorship. This is interesting, as it may explain the variation in productivity of domestic and market wells, where the water was reported to be ‘very hard’ (Robert et al., 1998), and the reason why mosquito actively avoids well with such conditions (Rim-Rukeh, 2013). The present study also provides explanation for an earlier observation (100% mortality) made in an attempt to rear mosquitoes using very hard water (unpublished data).

Effects of rearing-water pH and hardness level on emergence and longevity of Culex quinquefasciatus

The quality of a mosquito larval habitat determines the productivity of the habitat, in terms of number at emergence, post-emergence survivorship and longevity (Minakawa, Sonye, & Yan, 2005). Furthermore, the quality of the habitat is greatly affected by the availability of mineral element and food elements important for the development, which is chiefly determined by the pH and hardness conditions of the water habitat (Clark et al., 2004).

In the present study, pH values greatly affected the number of emergent adult mosquito, with significantly higher number of emerging female than male mosquitoes at all pH levels investigated. Significantly lower numbers of emergent were observed at pH values of 4.0 and 10.0. Post-emergence longevity was significantly affected by the pH value of water used for rearing. Those reared at pH 6.0 to 8.0 lived the longest than those from other regimens; the shortest-lived were from pH 4.0 and 10.0. Woodhill (1942) noted that at pH 3.6 to 4.2, the percentage of emergence was considerably reduced in Aedes aegypti.

Similarly, water hardness also affected the number of emergent imagoes, percentage emergence and post-emergence longevity of the species. For example, higher numbers of adult mosquitoes emerged from the soft water (0 mg/L CaCO3) regimen; beyond 90 mg/L CaCO3, the numbers of emergent significantly reduced, being the lowest at 210 mg/L CaCO3 (‘very hard’ water).

Generally, there were significantly higher numbers of female than male mosquitoes in all hardness regimens, with decreasing numbers of male and female as hardness condition increased. Similarly, hardness levels above 90 mg/L CaCO, significantly reduced adult longevity; mosquitoes reared at 210 mg/L CaCO3 lived the shortest.

The findings of this study are epidemiologically important as water pH and hardness conditions have played significant roles in determining the bionomics and population density of mosquitoes and the resultant frequency of human-mosquito contacts. This information, although subject to field experimentation and trials, can be applied in the development of cost-effective tactics towards the reduction of mosquito population density and hence degree of human-vector contact. For example, natural mosquito breeding habitats with an average of pH 7.0 may be more productive with mosquitoes than others; such habitats should be prioritized in terms of allocation of scarce resource for control. The reverse could be obtainable for mosquito habitats with extreme (acidic and alkaline) pH values. More so, if the hardness of a water body is known, it can give an indication of the level of productivity of that habitat, or perhaps be manipulated by the application of hardness-causing agents like lime water to it. Further, mosquito habitat with water hardness within the range of ‘very hard water’ may not support the proliferation of mosquitoes; therefore, effort should be channelled to other productive habitats with mild hardness condition.

Conclusion

The present study revealed significant effects of water pH and hardness conditions on developmental and vectorial attributes of Culex quinquefasciatus. Extreme pH values and increasing water hardness delayed development. While other entomological variables such as immature and adult survivorship, adult emergence success and post-emergence longevity were variously affected by the water conditions tested. The information generated in this study can also be incorporated in the development of cost-effective integrated mosquito management (IMM) protocol. However, field evaluation of the influence of prevailing physico-chemical parameters on biological fitness is advocated to verify these laboratory outcomes.