Evaluating the efficiency of syrphid fly larvae (Diptera) and khakibos extract (Tagetes minuta) against aphids (Brevicoryne brassicae)

October 29, 2014 matshidisolp

Abstract

Biological control is a technique of integrated pest management where by parasites, pathogens and or predators are used to control pest species. In addition, bio-pesticides, chemicals derived from plant extracts, are also used to increase the effectiveness of bio-control technique. Pests have a potential to affect economically important crops, Brassica oleracea is an example of such crop which is affected by cabbage aphid, Brassic brevicoryne. In the current study, an evaluation of the efficiency of syrphid fly larvae and khakibos extract is tested against the cabbage aphid. A total of 28 plants were exposed to 30 aphids each. Following aphid exposure, the plants were divided into four treatment groups; control (no treatment), natural enemy, plant extract, and water treated plants. Aphids were counted on each plant 13 days after the application of the treatment. The whole experiment was laboratory based. It was found that all treatments were non-significant under Kolmogorov-Smirnov and Shapiro-Wilk statistics. Control plants showed the highest number of insects, whereas those exposed to natural enemy showed least number of aphids. Our results support our null hypothesis. Tagetes minuta and syrphid larvae are effective biological control variables against aphids.

Introduction

Integrated Pest Management (IPM) is considered to be the paradigm technique to control insect pests, plant diseases, weeds and vertebrate pests (Kogan & Bajwa 1999). Different techniques and methodology are used by IPM, biological control being one of them. Biological control is defined as the action of parasites, predators, or pathogens in maintaining another organisms’ (the pest) population density at a lower average than would occur in their absence (DeBach 1964). Flint and Dreistadt (1998) describe biological control as a bio-effector method used to control pests on crops by involving an active human management role. The general principles of biological control include identifying potential biological agents, source for natural enemy, understanding their biology and their ecological services within pest-host ecosystems (Ye et.al 2013). Additionally, to increase the effectiveness of biological control, management procedures make use of botanical insecticides, chemicals based on plant extracts (Ye et.al 2013).

Aphids are known to be serious pest species in certain areas. In this study we look at the cabbage aphid, Brevicoryne brassicae Linnaneus (Aphididae). It is a species that originates from Europe, however now has a worldwide distribution (Kessing and Mau 1991). Cabbage aphid is an external pest on the leaves and flower stems of Brassica crops (Kessing and Mau 1991). The aphid feeds on phloem sap (Goggin 2007). One individual alone would not cause much damage to a whole plant; the damage is in the work-force of the whole population which leads to unsuitable crops. The rapid increase in population density of these aphids is a result of the short generation time and extremely high asexual fecundity (Kusnierczyk et.al 2008). However, there are means to control aphids. The host plant used in this study was Brassica oleracea Linnaneus (Brassicacea), a species of plant that includes food such as cultivars, including cabbage (Snogerup et.al 1990). Removal of sap of the plant by aphids weakens it and may sometimes lead to death (Ellis et.al 1998).

In the current study, extract was obtained from khakibos ,Tagetes minuta ,Linnaneus (Asteraceae) plants. T.minuta is a plant species that originates from South America and has been naturalized in South Africa (Holm et.al 1991). The strong-smelling essential oils of T. minuta have enabled it to be used for many purposes, including as a relish, laxative, diuretic, flavouring, insect repellent, stimulant and snuff (Holm et.al 1997). It widely known to have pesticidal properties due to the numerous secondary compounds it possesses (Soule 1993). The value extract is obtained from the plant through grinding, mixing with water and soap. The method is affordable to even small-scale farmers, and in addition relatively safe to both man and environment unlike most other pesticides.

Bio-control also makes use of natural enemies of targeted pests. Hoverflies (Diptera: Syrphidae) larvae are common predators of aphid in Brassica crops (Bugg et.al 2008), and on this basis, we selected syrphid fly larvae as a natural enemy. The adult hoverflies feed on pollen and nectar and are known to be efficient pollinators. The larval life stage is when they eat aphids, usually they feed at night, sucking larger aphids empty or eating smaller aphid’s whole (Bugg et.al 2008).

Methods and Materials

Study site and species

The study was conducted at the University of Pretoria, Department of Zoology and Entomology laboratory 3-1 (25.7536° S, 28.2297° E). The study was conducted for a period of two weeks (from 3^rd October 2014 to 17^th October).

The target pest species, B. brassicae L. was obtained from the botanical gardens of University of Pretoria and from Pretoria North. The host plants (B.oleracea) were grown in a controlled laboratory environment a few weeks before the experiment took place. Seedlings (± 8) were planted and covered with potting soil. The plants were grown to stage 13-14 (three to seven leaves unfolded) according to BBCH scale. Syrphid fly larvae were used as the natural enemy defence against the aphids.

Preparing the extract

Leaves (100g) from T. minuta were grinded into paste and mixed with one litre of water in a beaker. The beaker was then covered and kept at room temperature for 24hours. The mixture was then stirred and strained, followed by addition of few drops of soap (Teepol dishwashing liquid, AcornGroup).

Experimental design and treatments

A total of 28 plants were used in this experiment. Each plant was exposed to 30 aphids. Of the total, seven were treated with extract, another was treated with water, six were exposed to natural enemy (two larvae per plant), and seven were used as controls (just aphids only). In each treatment, the plant was sprayed twice (from upper angle and lower angle).

Completely randomized design was used to arrange the replicates of each treatment. Each replicate of each treatment was assigned a random number between one and seven. The treatments were then placed in a laboratory at temperature of 24ºC, natural humidity and photoperiod. After a period of 13 days, the aphids were counted on each plant.

Data analysis

Data was recorded using Microsoft Excel (Microsoft Office 2010). Using IBM SPSS (Statistica 12), box-whisker plots were constructed to check for outliers. In addition, Kolmogorov-Smirnov and Shapiro-Wilk statistics was used to analyse normality of data. The test for normality revealed significance for control treatment, therefore the data was log-transformed for mean of number of aphids after treatment. One-Way ANOVA and Post-Hoc LSD-multiple comparisons test were both ran to test for homogeneity of variances and test for multiple comparisons between treatments, respectively.

Results

All treatments were non-significant under Kolmogorov-Smirnov and Shapiro-Wilk statistics (P>0.05) (Appendix Table 1). Test of homogeneity of variances under One-Way ANOVA revealed no significance between treatments (Levene Statistics, F_{3, 23}=1.899, P> 0.05). However, ANOVA test revealed that there was a significance difference between the treatments (ANOVA, F_{3, 23}= 7.353, P<0.05). Multiple comparisons of transformed data revealed that there was a significant difference between the effectiveness of (1) control and natural enemy, (2) natural enemy and water, and (3) natural enemy and extract (Appendix Table 2). Using the data before transformation, we found that plants which were under no treatment (control) showed the highest number of aphids, followed by water and extract (Fig 1). Plants that were exposed to natural enemy showed the least number of aphids post-treatment.

Figure 1: Total number of aphids recorded on plants exposed to different treatments. All total of 28 plants were exposed to 30 aphids originally. A total of 28 plants were used in this experiment. Of the total, seven were treated with extract, another was treated with water, six were exposed to natural enemy (two larvae per plant), and seven were used as controls (just aphids only). Water and extract were sprayed twice to each plant.

Discussion

If the use of khakibos extract and presence of syrphid fly larvae enhances bio-control of cabbage aphid, then fewer aphids should be observed on the plants exposed to the natural enemy, and those that were treated with the extract, as compared to the control. The study did in fact find that plants exposed to natural enemy showed less number of aphids, and those treated with the extract. Our results indicate that syrphid fly larva is an effective natural enemy of cabbage aphid. Mahr et.al (1993) also found that syrphid fly larvae are effective predators in destroying cabbage aphids, especially small colonies. Furthermore, the colonies are usually favoured by humid weather, of which is unfavourable to aphids (Mahr et.al 1993).

Our results also indicate that the plant extract mixture from Tagets minuta is an effective bio-pesticide of cabbage aphids. Furthermore, T.minuta extracts have also been shown to significantly kill other insects such as beetles (Weaver et.al 1994), mosquitoes (Philogene et.al 1985) and armyworms (Rao et.al 2000). These studies have identified plant essential oils as the active ingredient that acts to kill larvae or prevent successful pupation from taking place (Dunkel et.al 2010). Extracts of these plants have also shown to repel these pests (USEPA 2012) giving it potential to be implemented in intercropping systems (Phoofolo et.al 2013).

The original aphids that were exposed to plants were just chosen randomly without taking into account the age structure or sex ratio. This might have affected the general population growth on plants that had fewer females. In addition, counting the aphid’s post-treatment, human error was eliminated by having two individuals count separately and taking the average of the two counts.

The objective of the current study was to evaluate the insecticidal effectiveness of crude extracts of T. minuta L. (Asteraceae) as well as that of syrphid fly larvae against cabbage aphids, B. brassicae. Our null hypothesis was that the extract and natural enemy exposed plants will have fewer aphids than that of control and water, and this was supported by the result of the study. The use of Tagetes minuta and syrphid fly larva should be well communicated to farmers, both small and large scale, to increase crop production that would be market suitable. Perhaps application of this extract can also be tested in other pests attacking Brassica crops and related species.

References

DeBach, P. 1964. The scope of biological control. p. 3-20. In Biological Control of Insect Pests and Weeds. Chapman and Hall Ltd., London. 844 pp

Dunkel, F.V., Jaronski, S.T., Sedlak, C.W., Meiler, S.U., Veo, K.D. 2010. Effects of steam-distilled shoot extract of Tagetes minuta (Asterales: Asteraceae) and Entomopathogeneic fungi on larval Tetanops myopaeformis. Environmental Entomology 39: 979-988

Ellis, P.R., Pink, D.A.C., Phelps, K., Jukes, P.L., Breeds, S.E., Pinnegar, A.E. 1998. Evaluation of a core collection of Brassica oleracea accessions for resistance to Brevicoryne brassicae, the cabbage aphid. Euphtica 103: 149-160

Ellis, S., Croxall, J. P., Cooper, J. 1998. Penguin conservation assessment and management plan: report from the workshop held 8-9 September 1996, Cape Town, South Africa. IUCN/SSC, Apple Valley, USA.

Flint, M.L. &Dreistadt, S.H. 1998.Natural enemies handbook: The illustrated guide to biological pest control. University of California.

Goggin, F.L. 2007. Plant-aphid interactions: molecular and ecological perspectives. Current Opinion Plant Biology 10(4): 399-408

Kessing JLM, Mau RFL. 1991. Cabbage aphid, Brevicoryne brassicae (Linnaeus). Crop Knowledge Master. Department of Entomology, Honolulu, Hawaii.

Kogan, M. and W. I. Bajwa. 1999. Integrated Pest Management: A global reality? An. Soc. Entomol. Brasil. 28:1-25.

Kusnierczyk, A., Winge, P.J.,, Stokmo, T.,Troczynska, J., Rossiter, J.T; Bones, A. 2008. Towards global understanding of plant defence against aphids – timing and dynamics of early Arabidopsis defence responses to cabbage aphid (Brevicoryne brassicae) attack. Plant, Cell and Environment. 31 (8).

Mahr, S., D. Mahr and J. Wyman. 1993. Biological control of insect pests of cabbage and other crucifers. N. Cent. Reg. Pub. 471; 54 pp.

Philogene, B.J.R., Arnason, J.T., Berg, C.W., Duval, F., Morand, P. 1985. Efficacy of the plant photoxin alphaterthienyl against Aedes intrudens and effects of nontarget organisms. Journal of Chemical Ecology 12: 893-898

Phoofolo, M.W., Mabaleha, S., Mekbib, S.B. 2013. Laboratory assessment of insecticidal properties of Tagetes minuta crude extracts against Brevicoryne brassicae on cabbage. Journal of Entomology and Nematology 5(6): 70-76

Rao, M.S., Pratibha, G., Korwar, G.R. 2000. Evaluation of aromatic oils against Helicoverpa armigera. Annual Plant Protection Sciences 8: 236-238

USEPA (United State Environmental Protection Agency). 2012. Biopesticides Registration Action Document- Tagetes Oil- PC Code: 176602. Accessed [http://www.epa.gov/pesticides/chem_search/reg_actions/registration/decision_PC-176602_22-Mar-12.pdf]

Weaver, D.K., Wells, C.D., Dunkel, F.V., Bertsch, W., Sing, S.E., Sriharan, S. 1994. Insecticidal activity of floral, foliar, and root extracts of Tagetes minuta (Asterales: Asteraceae) against adult Mexican bean weevils (Coleoptera: Bruchidae). Stored Product Entomology 90: 1678-1683

Ye, G.Y.,Xiao, Q., Chen, M., Chen, X., Yuan, Z., Stanley, D.W. & Hu, C. 2013.Tea: Biological control of insect and mite pests in China. Biological Control 9:14-21.

Appendix

Treat		Kolmogorov-Smirnov^a			Shapiro-Wilk
Treat		Statistic	df	Sig.	Statistic	df	Sig.
LogAphATMean	Control	.280	7	.104	.914	7	.427
	NE	.223	6	.200^*	.847	6	.148
	Extract	.186	7	.200^*	.906	7	.371
	Water	.204	7	.200^*	.886	7	.256

(I) Treat		Mean Difference (I-J)	Std. Error	Sig.	95% Confidence Interval
(I) Treat		Mean Difference (I-J)	Std. Error	Sig.	Lower Bound	Upper Bound
Control	NE	1.30482^*	.31377	.000	.6557	1.9539
	Extract	.45702	.30146	.143	-.1666	1.0806
	Water	.02820	.30146	.926	-.5954	.6518
NE	Control	-1.30482^*	.31377	.000	-1.9539	-.6557
	Extract	-.84780^*	.31377	.013	-1.4969	-.1987
	Water	-1.27661^*	.31377	.000	-1.9257	-.6275
Extract	Control	-.45702	.30146	.143	-1.0806	.1666
	NE	.84780^*	.31377	.013	.1987	1.4969
	Water	-.42881	.30146	.168	-1.0524	.1948
Water	Control	-.02820	.30146	.926	-.6518	.5954
	NE	1.27661^*	.31377	.000	.6275	1.9257
	Extract	.42881	.30146	.168	-.1948	1.0524

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Urine concentration and diluting abilities of Micaelamys namaquensis and Rhabdomys pumilio

October 14, 2014 matshidisolp

ZEN 361

Matshidiso Pitswane¹

UPID: 10294547

¹Department of Zoology & Entomology, University of Pretoria, Pretoria, 0002, South |Africa

Abstract

Mammalian rodents are distributed in different environments that include both mesic and arid. The concentrating ability of the kidney is one important function directly related to adaptation in arid conditions. In this experiment concentrating and diluting ability of two species of Southern Africa, Micaelamys namaquensis and Rhabdomys pumilio were studied. Animals were captured using Sherman traps. Both species were exposed to rat chow, sucrose, and control (rat chow and sucrose) diets differently. Volume of urine produced and osmolality were recorded. In M. namaquensis, we found that there was a significant difference in amount of urine produced by the species when fed different diets (F₂, ₁₅₄= 95.703, P<0.001). The same results were observed in R. pumilio, there was a significant difference in amount of urine produced by the species when fed different diets (F₂, ₁₅₄= 73.206, P<0.001). In M. namaquensis species there was a significant difference in osmolality across the different diets (F₂, ₁₁₅= 148.648, P<0.001).Similar results were obtained where R. pumilio were concerned, there was a significant difference in osmolality across the different diets (F₂, ₁₁₅= 136.485, P<0.001). Our results suggest that the two species have similar concentrating and diluting abilities regardless of their differences in habitat.

Introduction

Water is a limiting factor for many terrestrial rodent populations (Reaka and Armitage 1976. Moro and Bradshaw 1999a) and physiological adaptation of small rodents to arid conditions is mainly achieved through concentrating abilities of their kidney (Beuchhat 1996). The urine concentrating ability of a mammalian kidney is dependent on renal medullary size, a consequence of nephron morphology (Ntshotsho et al. 2004). Animals from more arid habitats generally have better urine concentrating ability than those found in mesic regions (Beuchat 1993, 1996). Immediate advantages associated with concentrating urine includes, water conservation and thus avoidance of dehydration which might lead to death.

In this experiment we look at two species, both from Southern Africa. Rhabdomys pumilio is one of the rodents that are known to pollinate Protea species (Heming and Nicolson 2002) and in essence ingesting a large amount of preformed water. These rodents feed on nectar amongst other things, and thus able to produce dilute urine (Heming and Nicolson 2002). R. pumilo are distributed in mesic regions (Pillay 2000). MIcaelamys namaquensis on the other hand occurs in more arid areas, the Namib Desert (Withers et al. 1980), and as suggested by Beuchat (1993, 1996) is probably capable of producing concentrated urine.

Diluting ability, unlike concentrating ability, has received little attention in the scientific world. In addition, most studies do not address hydration or dehydrating conditions and the time course of adjustment. This study aims to address whether good concentrating ability in a rodent will be correlated with good diluting ability or will it limit the diluting ability of the animal. Our null hypothesis is that there will be no difference in urine production and osmolality amongst the species between different diets.

Methods and Materials

Data collection

Five of each species were captured using Sherman traps baited with peanut butter, oats and golden syrup on Jonaskop (a mountain on the western end of the Riviersonderendberge, 33° 56’S 19° 31’E). The rats were kept in a UCT animal house, where they were fed rat chow and water, with natural light conditions and temperature variation of 17-22°C. The houses were standard rodent cages, with rat wheels, tunnels, and nest boxes, referred to in this paper as ‘normal housing’. The experimental rats were then kept in a constant environment room with 12h light: 12h dark (light from 07:00 to 19:00), with temperature of 20°C and humidity of 50%. In each normal house, a metabolism cage was set up to collect urine samples. For each species, 20 control rats were used. The controls were weighed initially, provided with 0.1M sucrose solution and rat chow, and the urine was collected under liquid paraffin every six hours for 24 hours, with level of sucrose solution recorded.

In the first trial of the experiment, five rats chosen at random were fed rat chow only and not given water. The remaining five animals were given access to 0.1M sucrose solution only. Urine samples were collected under liquid paraffin every five hours for overall 70 hours and frozen at -20°C for later analyses. All animals were weighed at the end of the experiment. In addition, the animals were returned to normal housing and allowed to rest for two weeks before the second trial. In the second trial, the same procedure as in the first trial was followed, with the following changes: animals which were fed 0.1M sucrose were fat rat chow and vice versa and there was no control period.

Data analysis

Volume of sucrose solution drunk and volume of urine were measured during the experiment. Osmolality concentration was measured on a Wescor 5500 vapour pressure osmometer, and samples of 10µwere used and the mean were recorded. Sodium (Na) and potassium (K) concentrations were also determined using an instrumentation Laboratory 243 flame photometer, and mean of the three values was then recorded. The data was analysed using IBM SPSS Statistics Version 22 (IBM Corporation). Volume of urine and osmolality data was log-transformed. Descriptive statistics, fixed effects, estimates and pairwise comparisons were recorded.

A total of 320 individual animals were used in this experiment, 160 of each species.

Results

In analysing urine production as function of different diets in M. namaquensis, we found that there was a significant difference in amount of urine produced by the species when fed different diets (F₂, ₁₅₄= 95.703, P<0.001) (Fig. 1). In other words, the differences in treatments results in differences in amount of urine produced within the same species. The same results were observed in R. pumilio, there was a significant difference in amount of urine produced by the species when fed different diets (F₂, ₁₅₄= 73.206, P<0.001) (Fig. 2). The average volume of urine produced by M. namaquensis was (mean±SD) 0.3506± 0.4227 ml/mol, whereas R. pumilio was 0.2964± 0.3823 ml/mol. In both species, the average volume of urine produced by animals on control and rat chow diet, lied below the total average, whereas that of animals on sucrose diet lied above the total average.

The second part of the experiment looked at osmolality concentration as a function of different diets. In M. namaquensis species there was a significant difference in osmolality across the different diets (F₂, ₁₁₅= 148.648, P<0.001) (Fig. 3). In other words, the differences in treatments results in differences in osmolality within the same species. Similar results were obtained where R. pumilio were concerned, there was a significant difference in osmolality across the different diets (F₂, ₁₁₅= 136.485, P<0.001) (Fig. 4).The average osmolality of M. namaquensis was 2.556 ± 0.745ml/mol and R. pumilio was 2.713 ± 0.803ml/mol. In both species, the average osmolality observed in animals on control and rat chow diets, lied above the total average whereas average of those animals on sucrose diet lied below the average.

A summary of pairwise comparisons analysed if each diet affected either urine or osmolality in a significantly different manner. In both urine volume and osmolality, the effect of rat chow and the control diet both differed significantly from the effects of sucrose, however we noted that the effects of rat chow and the control diets were not significantly different from each other (Fig 1, 2,3 and 4).

Discussion

Our results showed that different diets affect urine production in different ways. In both species, animals under sucrose diet showed a higher urine production that either animals under control and rat chow diets. Even though the two species inhabit different environments, the results suggest that they have similar osmoregulatory abilities. In another studied conducted by Ntshontsho et al. (2004), the authors found that urine excreted by control mice had lowest volume among other treatments. However, in this study we found that the lowest urine volume was associated with animals under rat chow diet. Possible reasons for the differences in this study and that of Ntshontsho et al. (2004) could be that our rat chow nutritional composition was similar to their control diet.

The results further showed that in both species the different diets affect osmolality in different ways. In both species, rat chow showed highest osmolality, followed by control and then sucrose diet. Osmolality is the concentration of a solution expressed as the total number of solute particles per kilogram. The data follows the prediction that high water intake results in dilute urine. In other words, animals under rat chow diet have a higher water intake than animals on either sucrose or control diet. We reject our null hypothesis that there will be no difference in urine production and osmolality amongst the species between different diets.

Our data suggest that differences in geographical distribution of M. namaquensis and R. pumilio in Southern Africa cannot be attributed to differences in osmoregulatory capacities. The two species show similar osmoregulatory abilities. The next question that this study aimed to address was whether good concentrating ability in a rodent will be correlated with good diluting ability or will it limit the diluting ability of the animal. MacFarland et al. (1969) and Sweeney et al. (1993) found that osmolality of solution is not necessarily a linear function of concentration unless the solution is very dilute. However, Tracy et al. (2002) found that dilution of synthetic urine of kangaroo rats introduced negligible errors in osmolality measurements. This could have been a factor in our data as well.

References

Beuchat, C. 1993. The scaling of concentrating ability in mammals. In: Brown, J.A., Balmer, R.J., (Eds.), Insight in Vertebrate Kidney Function. Cambridge University Press, Cambridge, pp 259-279

Beuchat, C.A. 1996. Structure and concentrating ability of mammalian kidney: correlations with habitat. American Journal of Physiology 271: 157-179

Ntshontsho, P., Van Aarde, R.J., Nicolson, S.W., Jackson, T.M. 2004. Renal physiology of two southern African Mastomys species (Redentia: Muridae): a salt-loading experiement to assess concentrating ability. Comparative Biochemistry and Physiology Part A 139: 441-447

Reakam, M.L., Armitage, K.B. 1976. The water economy of harvest mice from xeric and mesic environments. Physiological Zoology 49: 307-327

Pillay, N. 2000. Female mate preference and reproductive isolation in populations of the striped mouse Rhadbomys pumilio. Behaviour 137: 1731-1441

MacFarland, W.N., Wimsatt, W.A., 1969. Rebal function and its relation to the ecology of the vampire bat, Desmodus rotudus. Comparative Biochemistry and Physiology 25: 985-1006

Tracy, R.L., Walsberg, G.E. 2002. Kangaroo rats revisited: re-evaluating a classic case of desert survival. Oecologia 133: 449-457

Sweeney, T.E., Beuchat, C.A. 1993. Limitations of methods of osmometry: measuring the osmolality of biological fluids. American Journal of Physiology 264(3): 469-480

Figure 1: Log-transformed urine volume of three diet treatments: control, rat chow and sucrose of Micaelamys namaquensis. Bars represent mean average values of volume concentration of urine plotted with standard errors based on means. Rats that survived on sucrose diet showed an increased urine production than animals on rat chow and treatment.

Figure 2: Log-transformed urine volume of three diet treatments: control, rat chow and sucrose of Rhabdomys pumilio. Bars represent mean average values of volume concentration of urine plotted with standard errors based on means. Rats that survived on sucrose diet showed an increased urine production followed by animals on rat chow and then treatment.

Figure 3: Log-transformed osmolality concentration of three diet treatments: control, rat chow and sucrose of Micaelamys namaquensis. Bars represent mean average values of osmolality concentration plotted with standard errors based on means. Rats that survived on rat chow diet showed a higher osmolality, followed by control and then sucrose.

Figure 4: Log-transformed osmolality concentration of three diet treatments: control, rat chow and sucrose of Rhabdomys pumilio. Bars represent mean average values of osmolality concentration plotted with standard errors based on means. Rats that survived on rat chow diet showed a higher osmolality, followed by control and then sucrose.

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The effects of differing protein to carbohydrates ratios (P:C) on longevity & consumption of the female marula fruit fly, Ceratitis cosyra.

October 14, 2014October 14, 2014 matshidisolp

ZEN 361

Matshidiso Pitswane¹

UPID: 10294547

¹Department of Zoology & Entomology, University of Pretoria, Pretoria, 0002, South Africa

Abstract

It has been known for nearly a century that moderate restriction of dietary nutrients (dietary restriction) results in extended longevity and reduced reproduction in a broad range of animal species. To survive and reproduce, fruit flies need sugars as energy source and proteins to attain reproductive maturity and produce eggs. In this experiment we look at differing protein to carbohydrates ratios (P:C) on longevity & consumption of the female marula fruit fly, Ceratitis cosyra. We found that differing diet has no significant difference in mortality. However, it does affect consumption and oviposition. When female flies were given diet consisting of just sugar, they ate far less than when given a diet that contained protein, regardless of carbohydrates to protein ratio. The highest number of eggs were produced by the group of flies under the P:C 1:3 diet (16.971± 4.223) (data reported as mean ± standard deviation), followed by P:C 1:1 (13.765± 3.602), and lastly the lowest number of eggs produced were seen in P:C 0:1 diet (13.353± 3.062). In conclusion, we rejected our hypothesis that diet has no significant difference in consumption and oviposition.

Introduction

It has been known for nearly a century that moderate restriction of dietary nutrients (dietary restriction) results in extended longevity and reduced reproduction in a broad range of animal species (Mair & Dillin2008; Fontana et al. 2010; Nakagawa et al. 2012). In recent studies, it has been shown that certain species have trade-offs driven by protein, i.e. egg output by females is is enhanced by increased protein, however at the expense of reduced longevity (Lee et al. 2008; Maklakov et al. 2008; Je et al. 2009).In this study we used nutritional geometry methodology which looks at effects posed by diets composition on different life processes (Simpson and Raubenheimer 2007). This technique has been used in a couple of studies, including Lee et al. (2008) study that looked at demonstrating the key role of protein in mediating the trade-off between survival and reproduction in adult female flies.

In this study we look at the marula fruit fly, Ceratitis cosyra, a specialist that oviposits in fruits. Most of these flies transverse national boundaries and pose a serious threat to fruit production (Manrakan and Lux 2006). For example, assessment by the African Fruit Fly Initiative (AFFI) revealed that of the 1.9 million tons of mangoes produced annually in Africa, about 40% is lost to fruit fly infestation (Lux et.al 2003). Ceratitis cosyra has been reported to be the dominant species among other Ceratitis fruit fly species in attacking mango fruits in sub-Saharan Africa (Malio 1979, Javid 1986, Mukiama and Muraya 1994, Labuschagne et al. 1996, Lux et al. 2003).

The main diet of marula fruit fly is mainly from plants of families Anacardiaceae (mango, marula) Annonaceae (custard apple) and a small number of other families with soft, fleshy fruit. Carbohydrates and proteins are the main nutrients affecting reproductive performance and longevity in fruit flies. Caloric restriction (CR) has been widely accepted as mechanism explaining increased lifespan (LS) in organisms subjected to dietary restriction (DR), but recent studies investigating the role of nutrients have challenged the role of CR in extending longevity (Fanson et al. 2009). Manrakan and Lux (2006) reports that there has been no research on the contribution of natural food sources to adult longevity and fecundity of the other African tephiritid fruit flies, including C. cosyra species. This study also looks at whether survival is associated with diet treatment. The main objective of the present paper is to compare the effects of differing protein to carbohydrate (P:C) ratios on the longevity and egg production of the female marula fruit fly (C. cosyra). Our null hypothesis is that there will be no difference between the longevity and egg production of the female C. cosyra across the three different diets.

Methods and Materials

Source of study animals

All flies used in this experiment were obtained from a culture maintained in the Department of Zoology and Entomology, University of Pretoria. The culture had been maintained in a temperature-controlled laboratory (23-25°C) for over 21 generations with at least 100 adults in each generation. The adults had free access to a diet of granulated sucrose and hydrolysed yeast powder in separate dishes. Eggs were obtained from reproductively mature females through plastic cups covered with a layer of Parafilm (SPI Supplies) pierced with a number of holes through which females oviposit. Once collected the eggs were placed on an artificial diet based on carrot in which larvae went under development until ready to pupate. Third-instar larvae were permitted to migrate out of the larval diet into a shallow layer of sand in which they entered the pupal phase. Pupae were then shifted from the sand and placed in cages into which the adults emerged.

Fly treatment and housing

A total of 34 individuals carried out the experiment. Each person was given three cages, with one fly on each diet. Each cage consisted of two stacked 100mL clear plastic cups, with the bottom cup out of the inner cup. Mesh was used to cover the top of the inner cup. The oviposition substrate was placed on the floor of the intact outer cup as a parafilm-covered plastic lid (33mm diameter), which contained 4mL of an emulsion of fruit essence in distilled water (1:10 v/v). The parafilm was pierced 10 times with an entomological pin to encourage females to oviposit into the dish. The cages were kept a laboratory kept at 25°C and variable relative humidity.

Diets

Treatment diets consisted of varying ratios of hydrolysed casein (Peptro™) and sucrose dissolved in distilled water. The three diets used were: (1) sucrose only, (2) P:C (protein: carbohydrate) 1:3, and (3) P:C 1: 1. Diets and water were dispensed using separate pipette tips (Eppendorf 2-200µL) of diet or water. Pipette tips were capped with plasticine to decrease evaporation. Every day during the running period of the experiment, the tips were checked in order to ensure that diet and water was available to flies. In an event where either was empty, it was refilled and noted down. In addition, every five days, the diet and water that remained in the pipette tips was measured and replaced with fresh new tips which were filled with 200µL of water or appropriate diet. The nutrient intake was then calculated by measuring the length from pipette tip to liquid level using a ruler. The lengths were then converted to volumes by using a mathematical function (fitted using a regression equation: 0.0152x^2.5576 where x is the measurement taken by ruler in mm). A calibration curve was determined (Figure 1).

Evaporation controls

Each treatment diet had three controls to measure evaporative water loss and diet. These consisted of the same cage setup and protocol as treatment cages, only difference was that the control cages had no flies. Consumption values were corrected for evaporation.

Survival

The cages were inspected every day to record any mortality. On the day of mortality, the diet and water remaining was also recorded.

Data analysis

Mortality

A total of 99 flies were used in this study, 32 were under P:C ratio of 0:1, 34 were under P:C 1:3, and 33 were under P:C 1:1. Data was analysed using Microsoft Excel (Microsoft 2010). In calculating chi-square test, we found that less than 80% of the expected values were greater than 5, therefore we could not proceed with chi-square thus opted for Fisher’s exact test.

Consumption (measured in microliters)

The consumption data showed a normal distribution after analysis using IBM SPSS Statistics Version 22 (IBM Corporation). ANOVA pot-hoc analysis was performed.

Oviposition

Through the same program, we found that Oviposition did not fit a normal distribution curve, and therefore did not fir ANOVA model. Oviposition data was then analysed using Kruskal-Wallis test, which allows some assumptions of ANOVA model to be relaxed.

Results

Mortality

A total of 99 flies were used in this study, 32 were under P:C ratio of 0:1, 34 were under P:C 1:3, and 33 were under P:C 1:1. From the 99 flies that were used, 90 survived past the experimental period, and 9 died. Of the 9 that died, 1 was from P:C 1:1 diet, 2 were from P:C 1:3 diet, and the remaining from P:C 1:3. Fisher’s exact test revealed that there was no significant difference between the three diets in terms of mortality (Fisher’s exact test, df=2, P>0.05).

Consumption

There was a significant effect of diet on consumption by female C. cosyra (F_2,99= 6.611, P= 0.002). When female flies were given diet consisting of just sugar, they ate far less than when given a diet that contained protein, regardless of carbohydrates to protein ratio (Figure 2). Protein-carbohydrate ratio of 1:1 showed the highest consumption rate, followed by P:C 1:3, and lastly P:C 0:1 showing the lowest consumption volume. In looking at the effects between the diets, P:C 1:1 showed no significant difference in consumption volume as compared to P:C 1:3, and vice-versa. All the other combinations showed significant differences (Table 1).

Oviposition

The highest number of eggs were produced by the group of flies under the P:C 1:3 diet (16.971± 4.223) (data reported as mean ± standard deviation), followed by P:C 1:1 (13.765± 3.602), and lastly the lowest number of eggs produced were seen in P:C 0:1 diet (13.353± 3.062).

Discussion

To survive and reproduce, fruit flies need sugars as energy source and proteins to attain reproductive maturity and produce eggs (Webster and Stoffolano 1978). In our study we only lost less than 10% of the flies. Those that were lost could have been because of human error, lack of experience in handling of flies, and another possible reason is that the diet tips might have been placed in such a way that made it hard for the flies to access the diet contents. It is not suprising that diet did not have significant effect on mortality because one would think that survival is influenced by a lot of other factors, nutrients are probably more important than “diet” (Javid 1986). In this experiment we found that highest survival was observed in the sugar only diet compared to other diet ratios. In studies by Carey et al. 2002 the same results were obtained.

There is not much literature on consumption analysis of fruit flies and other insects in varying macronutrients. However, in our study we found that diet had a significant effect on consumption of the marula fruit fly. Those flies that were fed just sugar tend to eat a whole lot more than any of the other flies exposed to some protein supplement in their diets. This could be that sugar is not adequate enough by itself as a nutrient, and thus the flies ate more to supplement. In addition, we found that highest number of eggs were seen under P:C 1:3. Reasons for low number of eggs produced by female flies in other diets could be because some individuals were just infertile or non-viable, and perhaps the nutrients were not enough to provide the energy that goes with egg production.

In conclusion, we reject our hypothesis that diet has no significant difference in consumption and oviposition. We suggest that more studies should be conducted, in addition to supplement the methodology of Geometric framework.

References

Carey, J.R., Liedo, P., Harshman, L., Liu, X., Muller, H.G., Patridge, L. and Wang, J.L. 2002. Food pulses increases longevity and induce cyclical egg production in Mediterrenean fruit flies. Functional Ecology 16: 313-325.

Fanson, B.G., Weldon, C.W., Pe’rez-Staples, D., Simpson, S.J. and Taylor, P.W. 2009. Nutrients, not caloric restriction, extend life span in Queensland fruit flies (Bactrocera tryoni). Aging Cell 8:514-523.

Fontana, L., Partridge, L. & Longo, V. 2010. Extending healthy life span– yeast to humans. Science 328: 321–326.

Je, W.W., Carvalho, G.B., Zid, B.M., Mak, E.M., Brummel, T. & Benzer, S. 2009. Water- and nutrient-dependent effects of dietary restriction on Drosophila lifespan. Proceedings of the National Academy of Sciences United States of America 106: 18633–18637.

Lee, K.P., Simpson, S.J., Clissold, F.J., Brooks, R.C., Ballard, W.O., Taylor, P.W., Soran, N. & Raubenheimer, D. 2008. Lifespan and reproduction in Drosophila: new insights from nutritional geometry. Proceedings of the National Academy of Sciences 105: 2498–2503.

Mair, W. & Dillin, A. 2008. Aging and survival: the genetics of life extension by dietary restriction. Annual Review of Biochemistry 77: 727–754.

Maklakov, A.A., Simpson, S.J., Zajitschek, F., Hall, M.D., Dessmann, J., Clissold, F.J., Raubenheimer, D., Bonduriansky, R. & Brooks, R.C. 2008. Sex-specific fitness effects of nutrient intake on reproduction and lifespan. Current Biology 18: 1062–1066.

Malio, E. 1979. Observations on the mango fruit fly Ceratitis cosyra in the Coast Province, Kenya. Kenya Entomologist’s Newsletter 7.

Manrakhan, A and Lux, S.A. 2006. Contribution of natural sources to reproductive behaviour, fecundity and longevity of Ceratitis cosyra, C fasciventris and C. capitata (Dipetera: Tephritidae). Bulletin of Entomology Research 96: 256-268.

Nakagawa, S., Lagisz, M., Hector, K.L. & Spencer, H.G. 2012. Comparative and meta-analytic insights into life extension via dietary restriction. Aging Cell 11: 401–409.

Sentinella, A.T., Crean, A.J. and Bonduriansky, R.2013. Dietary protein mediates a trade-off between larval survival and the development of male secondary sexual traits. Functional Ecology 27: 1134-1144.

Simpson, S.J. & Raubenheimer, D. 2007. Caloric restriction and aging revisited: the need for a geometric analysis of the nutritional bases of aging. Journal of Gerontology, 62A: 701–713.

Figure 1: Consumption calibration curve. Fly consumption during the experiment was recorded in millimetres using a ruler. The lengths were then converted to volumes using a mathematical function relating length and volume for the pipette tip.

Figure 2: Mean consumption volume of female C. cosyra under three different diets varying protein and carbohydrates over a period of 15 days under 25°C. n=34

Figure 2: Mean oviposition (number of eggs produced ) of female C. cosyra under three different diets varying protein and carbohydrates over a period of 15 days under 25°C. n=34

Table 1: Multiple comparisons of different diets of varying protein: carbohydrate ratios against each other based on consumption volume of female C. cosyra. Sample size of 34.

DIET		Sig.
DIET		Sig.
P:C 0:1	P:C 1:1	.001
P:C 0:1	P:C 1:3	.013
P:C 1:1	P:C 0:1	.001
P:C 1:1	P:C 1:3	.327
P:C 1:3	P:C 0:1	.013
P:C 1:3	P:C 1:1	.327

Uncategorized

Desiccation resistance in Acheta domesticus crickets and the effects of size

October 14, 2014 matshidisolp

ZEN 361

Matshidiso Pitswane¹

UPID: 10294547

¹Department of Zoology & Entomology, University of Pretoria, Pretoria, 0002, South |Africa

Abstract

Desiccation in insects has always been viewed in correlation with water content, tolerance and regulation in harsh conditions, as well as temperature range of the environment. In this experiment, saturation deficit and water loss in the Mediterranean field crickets, Acheta domesticus, were studied. The experimental colony used included nymphs and adults varying in age and size, of which were kept at 28⁰C prior to experimentation. Initial mass of the crickets were recorded, and they were weighed every 24 hours. Each insect was kept in its own desiccation chamber which contained silica gel (drying agent). We assumed that, if the desiccation chamber is kept airtight and not opened very often, the humidity within the chamber stabilises at about 5%. Survival period and initial mass had no correlation (R²=0.02). Mass loss and mass loss rate showed moderate positive relationship with initial mass (R²=0.4 and 0.5 respectively). Our results suggest that initial body mass has no influence on survival, mass loss and mass loss rate increase with increasing initial body mass. The study can be improved by increasing sample size, separating the sexes as well as adults and nymphs. In addition, the methodology employed has limitations that might have affected final results.

Introduction

Temperature and water availability are considered to be among the most influential factors to the abundance and distribution of animals (Chown et.al 2002). Adaptations to arid or mesic environments are well known in many insects, showing selection can lead to a tight coupling between animals and their external environments. Physiological adaptations include high water contents or water storage capabilities in dry environments, lipid metabolism for the provision of water, tolerance of low body water contents, good regulation of haemolymph osmolality, and rapid water uptake after desiccation despite possible osmotic problems. At organismal level, desiccation resistance in insects is generally accomplished in three ways: by increasing body water content, by reducing rates of water loss, or by tolerating water loss of a greater proportion of body water (desiccation or dehydration tolerance) (Gibbs 1999).

In this study we look at desiccation resistance in the Mediterranean field crickets, Acheta domesticus (Linnaeus, 1758), and the effect of body size. This species inhabit semi-arid to arid environments (Huber et.al 1989). The crickets spend most of their life cycle outdoors, but will move indoors when conditions are unfavourable (Huber et.al 1989). Females generally have a larger body size than males. In addition, males are generally more active than females. The standard method of measuring desiccation resistance is to record the mass change of insects maintained in dry conditions (Chown et.al 2004). In this experiment, gravimetric technique was used, because it is both inexpensive and widely used, thus allowing for enough reference data. The entire experiment takes place in the lab under well monitored conditions. A physiological parameter, saturation deficit- an index of the evaporative power of air, is studied. The study also address the effects of body mass on desiccation resistance, differences between nymphs and adults, and finally the desiccation resistance of Acheta domesticus in comparison to other insects. In addition, the study reviews the limitations associated with gravimetric methodology of measuring desiccation resistance in insects.

Our hypotheses are: (1) adults will have a higher survival rate and will show stronger resistance than nymphs, and (2) females will show stronger resistance than males.

Methods and Materials

Data collection

The crickets used in this experiment are A. domesticus, from a colony that was maintained by the Department of Zoology & Entomology, University of Pretoria. The colony contained nymphs and adults varying in age and size. The insects were kept in a tank that was supplied with ProNutro™ and water for survival purposes. They were maintained at 28⁰C for 24h before the actual experimentation took place. The experiment was carried out by students, each with their own cricket selected randomly from the supplied colony. Each cricket was weighed in a small plastic container, using a balance scale (Model AR0640, Adventurer Ohaus Corp, New Jersey, USA), and the initial mass was recorded to three decimal places. Each cricket was then placed in its own desiccation chamber (600ml plastic cup with lid) over silica gel (drying agent). A piece of egg carton was provided as a refuge for each cricket, placed inside the desiccation chamber, and then the lid was firmly closed. We assumed that, if the desiccation chamber is airtight and is not opened very often, the humidity in the chamber stabilises at approximately 5%. All desiccation chambers were then labelled and kept at 25ºC. Each cricket was weighed at approximately daily intervals until death. At any point during the experiment, any moulted skin was removed before weighing occurred. Overall, 37 crickets were sampled.

Data analysis

The following data was collected for each cricket: initial mass at start of experiment, mortality date (if death occurred), survival period in hours, death mass, mass loss, and mass loss rate (mg/h). All mass values were recorded in mg. All data collected was manipulated using MsExcel (Microsoft, 2010). The following statistics were calculated: mean and standard error. Data was analysed using linear regression (R²), of which values range from 0 (no statistical correlation) and 1 (strong fit). All data is reported as mean ± SE, where SE refers to standard error.

Equations:

SE= STDEV/SQRT(n)

where SE is standard error, STDEV is standard deviation and SQRT means square root, and n is the total number of sample individuals.

Results

On average, the initial mass of a cricket was 253.6 ±17.9mg, with the lowest recorded at 44.3mg and the largest at 492.6mg. The average death mass was 127.6± 15.1mg ,average mass loss was 121.6 ± 12.6mg, and the average mass loss rate was 1.6 ± 0.2mg/h. The average death mass, average mass loss, and the average mass loss rate were calculated using n=28 because not all insects died at the conclusion of the experiment. Of all 37 crickets sampled, 28 died and only 9 survived. The time it took for 50% of the individuals to die (LT50) were 73 hours. From the whole sample colony, one individual only survived for 24hours and one individual survived for 97 hours, the rest lied between the two extremities.

Analysis of survival against initial mass revealed that there was no relationship between the two parameters (R²=0.02) (Fig.1). Initial mass and mass loss showed a moderate positive fit (R²=0.5) (Fig. 2). Similarly, initial mass and mass loss rate showed a moderate positive relationship (R²=0.4) (Fig. 3).

Discussion

Our results showed that survival period had no correlation with initial body mass in A. domesticus. Larger body mass is associated with higher body water content (Schulte-Hostedde et.al 2001). Better survival rates is seen in insects with a higher initial body water content than those with lower body water content during arid conditions (Gray and Bradley 2005). The time necessary to remove the amount of water required to kill an animal is lengthened in insects with higher amounts of water. Therefore, we had hypothesized that larger insects would survive longer than smaller insects. However, in our experiment the data did not support this prediction. The insects that survive beyond the experimental time frame showed variation in initial body mass with the lowest being 136.4mg. One reason could be that survival was measured in 24 hour intervals, perhaps if we measured in 3 hours interval we would have ended up with some form of a relationship.

Furthermore, the results showed that mass loss increases with increasing initial body mass (Fig. 2). Amount of water retained within animal’s body is affected by number of factors. Animal body water can vary with age, the older the individual, the higher its relative bone mass and thus the lower its body water content (Danks 2000). We can conclude from Danks (2000) that body water content is related to body mass. Data from this experiment suggest that larger insects tend to loss more body water than smaller insects. Similarly, mass loss rate increases with increasing initial body mass (Fig. 3).

In another study conducted by Matzkin et.al (2007), it was found that desert species were more desiccation tolerant, and in certain cases, variation was observed within the same species. In addition, the authors found that desiccation tolerance carried between sexes, females were more resistant than males.

Gravimetric methodology has been used extensively in measuring of water loss in arthropods (Ramlov and Lee 2000). Gravimetric method assumes that mass loss and water loss are equivalent (Wharton and Richards 1978). The method is susceptible to few errors including disruption of experimental temperatures and relative humidity (RH) during weighing (Hadley 1994).

References

Chown, S.L., Addo-Bediako, A., Gaston, K.J., 2002. Physiological variation in insects: large-scale patterns and their implications. Comparative Biochemistry& Physiology B 131: 587–602.

Gibbs, A.G. 1999. Laboratory selection for the comparative physiologist. Journal of Experimental Biology 202: 2709-2718.

Gray, E.M., Bradley, T.J. 2005. Physiology of desiccation resistance in Anopheles gambiae and Anopheles arabiensis. American Journal of Tropical Medicine and Hygiene 73 (3): 553–559.

Hadley, N. F. 1994. Water Relation of Terrestrial Arthropods. San Diego: Academic Press, Inc.

Huber, F., Moore, T.E, and Loher, W. 1989. Cricket behaviour and neurobiology. Cornell University Press, New York.

Matzkin, L.M., Watts, T.D. and Markow, T.A. 2007. Desiccation resistance in four Drosophila species: sex and population effects. Epub 1(5): 268-273

Ramløv, H. and Lee, R. E. 2000. Extreme resistance to desiccation in overwintering larvae of the gall fly Eurosta solidaginis (Diptera, Tephritidae). Journal of Experimental Biology 203:783 -789.

Wharton, G. W. and Richards, G. A. 1978. Water vapor exchange kinetics in insects and acarines. Annual Review Entomology 23:309 -328.

Desiccation resistance in Acheta domesticus crickets and the effects of size