Effect of Crushed Silver Cyprinid Rastrineobola Argentae Larval Diet on Pupae Eclosion and Mating Success of Male Anopheles arabiensis Mosquitoes  

Yugi J. O.1 , Ochanda O. H.2 , Mukabana W. R.2
1.Department of Biological Sciences, University of Kabianga, P. O. Box 2030-20200, Kericho, Kenya
2.School of Biological Sciences, University of Nairobi, P. O. Box 30197-00100, Nairobi, Kenya
Author    Correspondence author
Journal of Mosquito Research, 2016, Vol. 6, No. 12   doi: 10.5376/jmr.2016.06.0012
Received: 30 Mar., 2016    Accepted: 20 May, 2016    Published: 03 Jun., 2016
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Yugi J.O., Ochanda H., and Mukabana W.R., 2016, Effect of crushed silver cyprinid Rastrineobola argentae larval diet on pupae eclosion and mating success of male Anopheles arabiensis mosquitoes, Journal of Mosquito Research, 6(12): 1-6 (doi: 10.5376/jmr.2016.06.0012)

This study was undertaken to evaluate the effect of larval diet Rastrineobola argentae on pupae eclosion and mating success of adult Anopheles arabiensis Patton (Diptera: Culicidae). Rastrineobola argentae as a test diet and Tetramin® Baby as a comparative diet were fed to An. arabiensis larvae to generate adults raised exclusively on either of the two diets. Early pupae raised on the diets were placed in containers measuring 6 cm × 5.7 cm × 3.5 cm and assessed for pupae eclosion success. Emerged adults were sexed, transferred to holding cages and provided with 10% sugar solution. Ten three day old virgin male and female raised either on same or different diet types were paired in similar containers as pupae, given 10% sugar solution and left to mate. A wing was removed from experimental mosquitoes and measured to determine size. Spermathecae from every female was removed and examined for sperms. Diet type did not influence pupae eclosion success. Highest proportion of emerged (56%) and competitive (78%) males were associated with crushed silver cyprinid fish food. Female mosquito size influenced mating success (p=0.008) for Tetramin® Baby fish food raised mosquitoes and not crushed silver cyprinid fish (p=0.068) raised mosquitoes. Most females inseminated were raised from the same diet as the males. Diet type influenced mating success and size. High proportions of larger females were inseminated compared to smaller ones.
Anopheles arabiensis; Eclosion; Mating success; Rastrineobola argentae; Tetramin®


Malaria vector genetic control programmes such as the Sterile Insect Technique (SIT) rely on mass reared, genetically altered males that are released in large numbers to a target population. Efficient mass rearing procedures are integral components of procedures akin to SIT. However, mass rearing involves culturing mosquitoes in captivity a process which compromises fitness hence a need to test and determine fitness levels (Bargielowski et al., 2012) of such mosquitoes.


Different male insects employ different strategies to secure and mate with the females. For An. gambiae the males are reported to employ scramble type of strategy (Diabaté et al., 2011) over some swarm to secure mates. Body size (Voordouw and Koella, 2007; Maïga et al., 2012), age (Sawadogo et al., 2013) and genetics (Voordouw and Koella, 2007) dictate on which female is chosen while stamina dictated by the amount of energy reserves (Damiens et al., 2012; Maïgaa et al., 2014; Bellini et al., 2014) determines the mating success of the males. Captivity however, changes behaviour (Bartlett, 1984) of mosquitoes and impacts competitiveness in males as demonstrated in Culex tarsalis (Reisen et al., 1982).


However, the effect of captivity on male mating success may be modulated by correct diet formulation. This is because diet type does not only affect the physiology of mating (Ferguson et al., 2005; Takken, 2005) but also impart phenotypic characters that influence mating success (Howell and Knols, 2009). It was in respect to this observation that this study was undertaken to evaluate the effect of crushed silver cyprinid fish (Yugi et al., 2014) on pupae eclosion and mating success in male An. arabiensis mosquitoes. The effect of this diet type was compared to standard commercially larval rearing media TetraMin® Baby fish food. The experiments were conducted under a laboratory setting.


1 Results

1.1 Effects of diet type on An. arabiensis pupae eclosion success

The experiment was conducted for twenty days. It was found that eclosion success was not influenced by diet type. The highest proportion of emerged male mosquitoes was associated with pupae raised on crushed silver cyprinid fish diet (Table 1).



Table 1 Number of An. arabiensis pupae that eclosed for every 100 individuals raised on either crushed silver cyprinid fish or Tetramin® Baby fish in their larval stages and the proportions of adult mosquitoes emerging from the pupae. 1 Rows with number of pupae superscripted with the same letters don’t differ significantly; P represents the probability for the level of significance.


1.2 Effect of diet type on An. arabiensis male mating success

The experiment was conducted for six days. It was observed that male An. arabiensis mosquitoes raised on crushed silver cyprinid fish successfully inseminated female mosquitoes raised on a similar diet as them and not raised on diet different from them (Table 2). Insemination success of females by males raised on Tetramin® Baby fish food was significantly influenced by adult female mosquito size when males were paired with females raised on a similar diet (p=0.008) but not on a different diet (p=0.382). Mosquito size did not influence mating success for male mosquitoes raised on crushed silver cyprinid fish when paired with adult females raised on similar (p=0.068) or different (p=0.130) diet type.



Table 2 The percentage of female An. arabiensis mosquitoes inseminated when paired with males raised from the same as well as different diet type as the females. Sizes of female and male mosquitoes are shown in the form of mean plus standard error of means (Mean±SEM). 1 Row having mean male wing size superscripted with different letters indicates a significant influence of the mosquito size on adult male competitiveness; 2 Row having mean female wing size superscripted with different letters indicates a significant influence of the mosquito size on adult male competitiveness; 3 N is the number of test mosquitoes.


2 Discussion

This study demonstrates that there is differential contribution of different diet types to mosquito physiological processes. The highest proportion of emerging adult mosquitoes and inseminating male mosquitoes were associated with crushed silver cyprinid fish food. It was also observed that adult female size influenced mating success by the male mosquitoes.


Captivity has been demonstrated to alter insect behaviour especially male competitiveness for females (Bloem et al., 2004; Andreasen and Curtis, 2005; Allinghi et al., 2007). However, this quagmire has been found to be modulated by optimizing the quality of diet used to raise the adults as this compensate for the altered physiological demeanor due to confinement or genetic manipulation (Kassim et al., 2012; Puggioli et al., 2013).


Earlier it had been demonstrated that the dietary components of crushed silver cyprinid fish food influenced adult size and pupae eclosion (Yugi et al., 2014) and in this study it influenced the sex of emerging mosquitoes as the highest number of emerging adults were associated with it. Additionally, it was also associated with the highest number of mating males showing that it had a significant impact on the physiology of the mosquitoes. In this regard, the findings were similar to those observed for Aedes Albopictus (Yoshioka et al., 2012; Puggioli et al., 2013), An. stephensi (Khan et al., 2013) and An. arabiensis (Damiens et al., 2012; Hood-Nowotny et al., 2012).


It is not clear why improved insemination occurred when mating couples were raised on similar diet type as opposed to when the couples were raised on different diet. However it has been demonstrated using butterflies (Boggs and Freeman, 2005) and yellow dung fly (Reim and Teuschl, 2006) that larval diet types boosts reproductive energy and by extension fitness. This indeed was the explanation for the observation made on male An. arabiensis raised on crushed silver cyprinid fish food as well as Tetramin® Baby fish food.


Several studies have tried to investigate the role of body size in sexual selection and showed that male mate selection, and preference for larger females, had an influence on the genetic quality of offspring (Okanda et al., 2002). Accordingly in conditions where large numbers of males must compete for a smaller number of females, the largest females are more likely to become inseminated and in turn produce large egg batches (Okanda et al., 2002).


In this study, female mosquito size influenced mating success especially when the mating pair were fed on similar diet in their larval stages. However, while the benefit of large body size is clear in females, it is not clear how variation in size can influence An. arabiensis males mating success. This study did not test on male competitiveness and therefore it is hard to speculate on how male size could have influenced mating success.


In conclusion the current study shows that pupae arising from crushed silver cyprinid fish larval diet rapidly transformed to adults and emerged females relatively larger than those arising from the standard larval diet, Tetramin® Baby fish food. This demonstrates that with refinement and supplement addition, crushed silver cyprinid fish food should be an alternative larval diet for mass generation of mosquitoes for laboratory as well as procedures akin to the sterile insect techniques (SIT).


3 Materials and Methods

3.1 Study site and mosquito source

This study was carried out at the laboratories and insectaries of the School of Biological Sciences, University of Nairobi. The Anopheles arabiensis Patton mosquitoes used for the study were sourced from the International Atomic Energy Agency (Seibersdorf laboratories) in Vienna, Austria. The strain was originally from Dongola in northern Sudan.


3.2 Larval diet preparation

One kilogram of R. argentea also known as sardine in English was bought from a local market, oven dried to a constant temperature, crushed into powder using a food blender and then put in a glass vial. Tetramin® Baby fish food was obtained from I.A.E.A., Seibersdorf laboratories in the Netherlands. The two food types were kept under refrigeration at 4ºC (Yugi et al., 2014). The commercial Tetramin® Baby fish food was used as a positive control.


3.3 Mosquito culture

The mosquitoes used in the studies were from the F5 generation onwards. The mosquitoes were reared following standard techniques (Amalraj et al., 2005). The larvae in each tray were fed thrice daily, at 9.00, 13.00 and 17.00 hours. Each tray received an approximate of 3 milligrams of the respective larval diet type each day. The adults were offered 10% sugar solution soaked in cotton pads placed on top of the cages, as a source of energy. The energy sources were offered on a daily basis. Two days after emergence, the females however were offered cattle blood collected from an abattoir and mixed with EDTA to prevent coagulation. The blood was provided via Hemotek® membrane feeding apparatus. On the second day after the blood meal, an oviposition dish was placed in the adult-holding cages for the collection of eggs. Mosquito egg collection, dispensing, larvae feeding, changing and the holding conditions were done as described in details elsewhere (Yugi et al., 2014). The conditions within the insectaries were maintained at temperatures of 27-30ºC relative humidity of 70-80% and photoperiod of 12 hours of light alternated with 12 hours of darkness.


3.4 Assaying to test effects of diet type on An. arabiensis pupae eclosion success

An informal ‘after-only with control’ experimental design (Kothari, 2004) was used. The design was to investigate the effect of crushed silver cyprinid fish on the ability of An. arabiensis pupae to emerge into adults. The experiment was conducted in twenty days. Each day ten early pupae raised on crushed silver cyprinid fish in their larval stages were placed individually in ten plastic containers measuring 6 cm × 5.7 cm × 3.5 cm with 10 mL of clean water and mouth secured with netting material to prevent escape of emerging adults. The procedure was repeated for pupae raised on Tetramin® Baby fish food. Cotton wool soaked in a solution of 10% sugar solution was placed on top of each of the netting material to provide nutrition for emerged adults. The experiments were started at 16.00 hours each day and the set ups left to stand in the insectary until all adults emerged or pupae were unable to give rise to adults. The experiment was replicated five times. The number of adults emerging from the pupae per treatment were recorded and the sex determined by looking at the nature of the antennae [male mosquitoes have bushy antennas (plumose) while female antennae are less bushy (pilose)].


3.5 Assaying for male mating success in An. arabiensis

A 1:1 in a 2 × 2 simple factorial design (Kothari, 2004) was used to assay the level of male mating success. The experiment was conducted for six days. Each day ten 3 day old virgin male and female mosquitoes raised on one of two larval diet types’ crushed silver cyprinid fish or Tetramin® Baby fish food were used. Ten male - female pairs were assessed each day in each category for six days. Each day, four combinations of adult male-female pairs were used. Each combination contained a pair of male-female adult mosquitoes placed in plastic containers measuring 6 cm × 5.7 cm × 3.5 cm with mouth covered with netting material. The combinations were as illustrated (Table 3). The male and female couples were provided with 10% sugar solution for nutrition. The experiment was replicated six times. The experiment was started at 18.00 hours every day and stopped at 8.00 hours the following day. At the end of the experiment, all mosquitoes were killed and a wing removed and measured to determine mosquito’s size.



Table 3  A 2 × 2 simple factorial design showing mate pairings of adult male and female mosquitoes raised on crushed silver cyprinid fish or TetraMin® Baby fish food. N is the number of test mosquitoes.


3.6 Examining the spermathecae for sperms

Each of the female mosquito was placed in phosphate buffered saline prepared as described (Cheesbrough, 1992) and dissected under a dissecting microscope (Leico Zoom 2000) to remove spermathecae. This was done by grasping the terminalia of the female mosquitoes and pulling away slowly using a fine tip needle. Once located the spermatheca was isolated, removed, a cover slip placed over it and observed under 1000× magnification. The spermatheca was judged as positive (inseminated) if it appeared opaque with telltale wriggling sperms within it or negative (no sperm present in spermatheca) if it appeared transparent with no wriggling sperms within it.


3.7 Statistical analysis

The data was organized in excel sheets and tested for conformation to the assumptions of normality. Proportion of emerged adults (male and female), inseminated female mosquitoes were analyzed as a function of mosquito size (based on wing length), diet type, level of success of insemination of female mosquitoes and all possible interaction thereof as predictors. Regression procedure and Pearson’s Correlation statistics (Field, 2000) was used to determine the statistical effect of the diet on the parameters. All such analysis was done using the Statistical Package for Social Scientists (SPSS for windows version 11.5). All such effects were considered significant at P<0.05.


Authors’ contribution

YJO did the experiments, collected, analyzed and interpreted the data, wrote and edited the manuscript. HO and MWR provided supervisory and guidance during experimentation read and corrected the manuscript. MWR sourced for the funds.



I thank Jessicah Anyango, Charlotte Awuor, Dalton Ochieng’ Fred Okumu and Sheila Barasa for helping breed the mosquitoes. I also thank International Atomic Energy Agency (I.A.E.A., Seibersdorf laboratories) for the provision of An. arabiensis mosquito eggs and funds to support this project (Research grant contract # KEN-13291).



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