## Introduction

Musca domestica Linnaeus is an economic pest of animal agriculture and public health which grows rapidly in unhygienic environmental conditions. It is not only a source of nuisance but also plays a role in disease transmissions through its rapid expansion and forcing the affected communities to rely on the heavy use of various insecticides1. This practice has been adopted worldwide with the resulting evolution of resistance in M. domestica against a number of insecticides2,3,4,5,6. As a consequence, the affected people shift from the recommended dose to over-dosage of insecticides with the ultimate negative impact on the environment and public health7. Therefore, the search for new insecticides to manage resistant insects and to develop resistance management strategies is of prime importance8. Among these relatively new insecticides, spinosad (derivative of a soil actinomycete Saccharopolyspora spinosa), has been considered safe due to very low toxicity towards mammals and other non-target organisms9,10,11,12. However, different insect pests like Aedes albopictus (Skuse)13, Drosophila melanogaster Meigen14, Plutella xylostella L.15, Helicoverpa armigera (Hübner)16, Tribolium castaneum17 and M. domestica6,18 have developed resistance against spinosad in some parts of the world.

There are some important factors which contribute to an insect pest developing resistance to a particular insecticide. These factors include the performance of biological parameters of a species in the presence of insecticide selection pressure, fitness costs, frequency of resistance allele(s), pest management practices and population dynamics19,20. Among these factors, fitness costs and the performance of biological parameters associated with resistance to a particular insecticide seem to vary considerably among species and insecticides21. The fitness costs i.e., lengthened developmental time, reduced fecundity and survival, have been widely assumed to be linked with mutations that confer insecticide resistance22. Generally speaking, fitness costs can be enhanced under stressful environments like the presence of insecticide selection pressure23. The poor performance of biological parameters and fitness costs due to insecticide resistance contribute to limiting an increase in resistance alleles24. Similarly, in the presence of these factors in a population, the stability of resistance allele(s), and subsequent transmissions to the next generations could be prevented by integrated management practices along with the wise use of insecticides24,25.

Previously, we have reported low levels of resistance to spinosad in M. domestica from Pakistan26. Thereafter, in order to understand the nature of resistance, we have reported on the inheritance and preliminary mechanism of spinosad resistance in M. domestica under laboratory selections27. The resistance was inherited autosomally, incompletely dominant and governed by more than one gene. In addition, absence of metabolic mechanism of resistance was reported in the resistant strain suggesting the possibility of an altered target site mechanism18. However, resistance development to spinosad was unstable and declined rapidly when spinosad selection pressure was lifted, thus forming the basis of a hypothesis that decline in resistance could be associated with fitness costs. There are a number of reported cases where high fitness costs were linked with spinosad resistance development in different insect species28,29,30,31. However, this information is sparse in the case of M. domestica. Studies on fitness costs in resistant populations are important for devising an effective resistance management strategy, and to prevent the misuse of insecticides32. Such studies ultimately help to minimize environmental pollution and negative impacts on public health. Therefore, the purpose of the present study was to determine the effects of spinosad resistance on different fitness/biological parameters of M. domestica. For this purpose, a near isogenic strain of M. domestica resistant to spinosad was established since such an approach is generally regarded asthe best for fitness assessments33.

## Results

The median lethal concentrations (LC50s) for Spin-UNSEL and Spin-SEL strains were 1.93 and 122.56 µg/ml, respectively. The Spin-SEL strain showed 155.14 fold resistance ratio (RR) to spinosad when compared with the Lab-susceptible strain27 (Table 1). In the process of isolating the near isogenic line with spinosad resistance, the RR values in the BCnF1 progeny decreased significantly after backcrossing with the susceptible recurrent parent; however, these values increased in the BCnF2 progeny after self-breeding. The LC50 value increased to 143.86 µg/ml in the BC5F2 progeny, and did not fluctuate in the next progenies (BC5F3, or BC5F4) based on the overlapping 95% CIs, suggesting that the near isogenic line (Spin-R) with spinosad resistance was established (Table 1).

The comparison of various biological parameters of Spin-UNSEL, Spin-SEL and Spin-R strains revealed significant differences (Table 2), that might reflect the presence of fitness costs in M. domestica. For instance, the survival of the Spin-R strain at the larval stage was significantly lower (65 ± 4.86%) compared with the Spin-SEL (71.33 ± 2.07%) and Spin-UNSEL (87.67 ± 1.80%) strains (F = 33.6; df = 2,12; p < 0.01). The larval stage took more time to complete in the Spin-R strain (6.20 ± 0.57days) as compared with the Spin-SEL (5.20 ± 0.12days) and Spin-UNSEL (4.40 ± 0.19days) strains (F = 21.2; df = 2,12; p < 0.05). Relatively heavier pupae were observed in the case of Spin-UNSEL (19.90 ± 0.56 mg) compared with the Spin-SEL (17.54 ± 0.21 mg) and Spin-R (16.08 ± 0.32 mg) strains (F = 29.9; df = 2,12; p < 0.01). The pupae of the Spin-UNSEL strain developed faster (4.80 ± 0.27days) than those of the Spin-SEL (5.70 ± 0.27days) and Spin-R (6.90 ± 0.42days) strains. In short, the total development time (from egg hatching to adult formation) lengthened in case of the Spin-R strain (13.30 ± 0.57days) compared with the Spin-SEL (12.20 ± 0.27days) and Spin-UNSEL (10.20 ± 0.45days) strains (F = 12.35; df = 2,12; p < 0.001).

Besides developmental time, other biological parameters were also significantly different between the studied strains of M. domestica. For example, 82.67% pupae of the Spin-UNSEL strain were successfully eclosed to adults as compared to 75 and 65% in the case of Spin-SEL and Spin-R pupae, respectively (F = 27.8; df = 2,12; p < 0.05). Comparison of the resultant adults revealed a relatively higher number of female M. domestica in the Spin-UNSEL strain (49.98 ± 3.13%) followed by the Spin-R and Spin-SEL strains (F = 4.41; df = 2,12; p < 0.05). Moreover, resultant female M. domestica of the Spin-UNSEL strain produced a higher number of eggs in their lifetime (340.80 ± 11.37) compared with the Spin-SEL and Spin-R strains (F = 545; df = 2,12; p < 0.001), and the proportion of egg hatching also showed the same trend. Regarding survival at the adult stage, the adults of the Spin-UNSEL strain lived significantly longer compared with the Spin-SEL and Spin-R strains (Table 2).

The comparison of fitness parameters including the intrinsic rate of natural increase (rm), biotic potential (bp), mean relative growth rate (MRGR), net reproductive rate (Ro), and relative fitness also revealed significant differences among all the strains. The values of all of the above fitness parameters were significantly higher in the Spin-UNSEL strain than those of the Spin-SEL and Spin-R strains (F = 198; df = 2,12; p < 0.01 for rm, F = 8.97; df = 2,12; p < 0.05 for MRGR, F = 120; df = 2,12; p < 0.001 for bp, and F = 202; df = 2,12; p < 0.001 for Ro, Table 2).

## Discussion

Environmental contamination resulting from the excessive use of pesticides in agriculture and health sectors can exert strong selection pressure on the exposed populations. Resultantly, survival of the fittest phenotypes with the ability to combat contaminations may arise as a consequence of evolution of resistance to the environmental contaminants1. Such directional evolutions in the selected populations are usually expected to have detrimental effects on fitness in the absence of environmental contaminants or selection pressure, thus constrain the evolution of resistance34. For example, worker honey bees reared in a pesticide-contaminated brood were found with lower survivorship, lengthened developmental time and higher brood mortality rates compared with bees reared in relatively uncontaminated brood, leading to the assumption that the bees in the contaminated brood were less fit and unable to evolve resistance against pesticides35.

The present study was based on the effects of spinosad resistance development on biological parameters in M. domestica. In our previous work27, the Spin-SEL strain showed rapid development of resistance to spinosad under laboratory selections, but resistance was unstable when the selected strain was reared without spinosad. Based on the unstable nature of resistance, it was hypothesized that fitness costs were associated with resistance in the selected strain of M. domestica. In the present study a near isogenic Spin-R strain was established to confirm the presence of fitness costs and the results have further strengthened the above hypothesis. The comparison of biological parameters of resistant and susceptible strains is a logical approach to evaluate fitness costs; however, misinterpretation of the fitness data may occur owing to genetic background variations between strains36. Therefore, in the present study, the near isogenic line of M. domestica resistant to spinosad was established. Theoretically speaking, the genetic variations between recurrent parental strain and the near isogenic line would only be in a limited number of genes, including the selected gene of interest37.

In the present work, the Spin-R strain was compared with Spin-SEL and Spin-UNSEL in terms of development from the egg stage to the adult formation, and in the performance of subsequent adults (fecundity, egg hatching, male to female ratio and survival). The results revealed significant differences in the performance of all the studied biological parameters among the studied strains which showed fitness costs associated with spinosad resistance. The Spin-R strain took significantly more time at the larval stage, which did not translate into heavier pupae compared with Spin-SEL and Spin-UNSEL. The Spin-R strain took more time to develop into adults as compared with Spin-SEL and Spin-UNSEL. Moreover, fecundity, egg hatching and survival at adulthood also decreased significantly in the Spin-R strain. Therefore, spinosad resistance in the Spin-R strain of M. domestica probably imposes direct fitness costs thus suggesting a natural compromise in the distribution of resources between biological parameters and resistance development allele(s) of the selected strain38.

Insecticide resistance mechanisms (e.g., target site or metabolic) could be responsible for inducing fitness costs in the Spin-R strain of M. domestica. Previously, it has been reported in different insect species that exon skipping39, point mutations40, or the production of truncated proteins41 might be responsible for spinosad resistance in the nicotinic acetylcholine receptor site and associated fitness costs. Similarly, for metabolic resistance mechanism, if the production of detoxifying enzyme is costly, then resistant individuals would not produce such enzymes in the absence of insecticide selection pressure22. Overexpression of cytochrome P450 genes has been reported in a spinosad resistant strain (791spin)42 and a Danish field strain (791a)43 of M. domestica. However, a high level of spinosad resistance in of M. domestica are not associated with P450 mediated mechanisms18,44. Similarly, resistance to spinosad in our Spin-SEL strain of M. domestica was unchanged upon pretreatment with an enzyme inhibitor PBO which pointed to the likely absence of P450 metabolic resistance mechanism27. However, further studies are needed to confirm the exact mechanism of spinosad resistance in the Spin-SEL strain and associated fitness costs.

The values of fitness parameters like MRGR, bp, rm and Ro of the Spin-SEL strain were also significantly reduced. These parameters indicate the potential of a certain population to increase under given environmental conditions49,50. The reduced rates of all these parameters in the Spin-R strain clearly demonstrate the presence of the fitness costs phenomenon. In this case, fitness costs is advantageous in terms of managing insecticide resistance since the removal of selection pressure will likely cause a decrease in the number of resistance allele(s)29,38, and ultimately reversion of insecticide resistance. This has already been reported in our previous work in which the Spin-SEL strain showed reversion of spinosad resistance when the selection pressure was lifted27.

In conclusion, the data of the present study demonstrate that fitness costs are most probably associated with spinosad resistance in the Spin-R strain of M. domestica. We found significantly reduced rates of adult eclosion, fecundity, egg hatching, survival, and lengthened developmental time in the Spin-R strain. Moreover, the values of different fitness parameters like biotic potential, mean relative growth rate, intrinsic rate of natural increase, and net reproductive rate, were also significantly reduced in the Spin-R strain, which reflect fitness costs most probably linked with spinosad resistance. The presence of fitness costs suggests likely instability of resistance to spinosad in M. domestica which can be reverted by relaxing spinosad selection pressure and rotation with alternate insecticides. The wise use of insecticides will ultimately help to manage resistance in this pest and minimize environmental pollution. Therefore, combined with our previous findings27, spinosad resistance in M. domestica could be managed by rotational use of insecticides. This will help to manage resistant insects effectively and minimize environmental pollution.

## Materials and Methods

### Musca domestica strains

A field strain of M. domestica was collected from Lahore (31° 32′59 N; 74° 20′37 E) and divided into two sub strains. One sub strain was kept unselected and reared up to 10 generations without exposure to any insecticide, and this strain was designated as “Spin-UNSEL”. The other sub strain was cultured under spinosad selection pressure for 10 consecutive generations and resulted in a 155 fold resistance development compared with the laboratory susceptible strain (Lab-susceptible)27. This sub strain was designated as “Spin-SEL”. A near isogenic line (Spin-R) with spinosad resistance was established following the methodology of Horikoshi, et al.36. In short, the Spin-R strain was derived by repeatedly backcrossing Spin-SEL females with Spin-UNSEL males. The offspring of the backcrosses were referred to as the BCnF1 progeny (n = number of backcrosses). The self-bred BCnF1 progeny yielded BCnF2 progeny which was selected with spinosad using the concentration level to cause 70% mortality (i.e., LC70), and then the surviving females were backcrossed to Spin-UNSEL males. The detailed methodology for selection experiment and bioassays has already been reported previously27. The selection bioassays and backcrossing were completed when the LC50 values of the self-bred progenies BCnF2, BCnF3, and BCnF4 had become stable. All the strains were maintained under laboratory conditions as described previously26,51.

### Survival and development of Spin-UNSEL, Spin-SEL and Spin-R strains

Survival and development was checked by following the work of Khan, et al.50 with a few modifications. Briefly, batches of 60 first instar larvae (<12 h old) of Spin-UNSEL, Spin-SEL and Spin-R strains were maintained on larval media under the above laboratory conditions. These batches were replicated five times to study fitness parameters, and the total number of larvae studied per strain was 300. The growth and survival of the tested larvae were checked twice daily. The parameters recorded were: duration of the larval (1st to 3rd instar) and pupal stages, survival at larval and pupal stages, and total developmental time from hatching to adult formation.

### Fecundity, egg hatching and survival at adulthood

To compare the number of eggs laid, egg hatching and survival at the adulthood stage, five pairs of all the strains (<24 h old) each were kept in separate wooden mesh cages (30 × 30 × 30 cm). The cages were provided with adult food, and larval medium for egg laying and immature development. The cages were kept under the same laboratory conditions as stated earlier. The cages were observed twice daily to count the number of eggs laid, eggs hatched and survival of the adults till the mortality of each pair.

### Fitness parameters

In order to calculate the mean relative growth rate (MRGR), 1st instar larvae (n = 50) of all the strains were taken from their respective cages, weighed, and divided into five equal batches. Each batch was reared separately in a 250 ml glass beaker containing 100 g larval medium. After the completion of the larval stage, pupae were removed and weighed to calculate MRGR according to the following formula52,53:

$${\rm{MRGR}}=\frac{[\,\mathrm{ln}\,.({\rm{pupal}}\,{\rm{weight}})-\,\mathrm{ln}\,.({\rm{initial}}\,{\rm{larval}}\,{\rm{weight}})]}{{\rm{Time}}\,{\rm{from}}\,{\rm{the}}\,{\rm{larval}}\,{\rm{stage}}\,{\rm{to}}\,{\rm{the}}\,{\rm{pupal}}\,{\rm{stage}}\,({\rm{T}})}$$
(1)

Net reproductive rate (Ro), intrinsic rate of natural increase (rm) and biotic potential (bp) were calculated as described previously53.

Biotic potential was determined by dividing the log fecundity with total developmental time50,53, and relative fitness (w) was calculated by dividing Ro of Spin-SEL or Spin-R strain with Ro of Spin-UNSEL strain.

To compare each biological parameter of the Spin-SEL, Spin-UNSEL and Spin-R strain, the data were analyzed by the multivariate analysis of variance (MANOVA) and means were compared by Tukey’s HSD test.

### Ethical statement

The study/bioassay protocols used against M. domestica were performed according to the standard guidelines and regulations and approved by the institutional research project evaluation committee.