Abstract:

Objective:

To analyze the transcription pattern of neuropeptides in the ontogeny of a malaria vector, the mosquito Anopheles albimanus.

Materials and methods:

The transcription pattern of Crustacean CardioActive peptide (CCAP), corazonin, Ecdysis Triggering Hormone (ETH), allatostatin-A, orcokinin, Insulin Like Peptide 2 (ILP2), Insulin Like Peptide 5 (ILP5) and bursicon was evaluated using qPCR on larvae (1^st - 4^th instar), pupae and adult mosquitoes.

Results:

Unlike in other insects, transcripts of CCAP (70.8%), ETH (60.2%) and corazonin (76.5%) were expressed in 4^th instar larvae, probably because these three neuropeptides are associated with the beginning of ecdysis. The neuropeptide ILP2 showed higher transcription levels in other stages and orcokinin decreased during the development of the mosquito.

Conclusion:

The CCAP, corazonin and ETH neuropeptides are potential targets for the design of control strategies aimed at disrupting An. albiamnus larval development.

Keywords:
gene expression; neuropeptides; Anopheles; insect

Resumen:

Objetivo:

Describir la expresión de neuropéptidos durante la ontogenia del mosquito vector de la malaria Anopheles albimanus.

Material y métodos:

Se midió la expresión de CCAP, corazonina, ETH, allatostatina, orcokinina, ILP2, ILP5 y bursicon en larvas de primer (2mm), segundo (4mm), tercer (5mm) y cuarto (6mm) estadio, pupas y mosquitos adultos, mediante qPCR.

Resultados.

A diferencia de otros insectos en donde, CCAP, corazonina y ETH se expresan principalmente en estadios pupales, en An. albimanus se expresaron mayoritariamente en larvas de cuarto estadio, CCAP tuvo 70.8% de expresión relativa, corazonina 76.5% y ETH 60.2%. ILP2 fue el neuropéptido que más se expresó en el primer, segundo y tercer estadio y orcokinina disminuyó durante el desarrollo del mosquito.

Conclusión.

Los péptidos estudiados se expresaron en todos los estadios de desarrollo del mosquito. Sin embargo, su expresión varió en cada uno de ellos. Los neuropéptidos CCAP, corazonina y ETH, que son esenciales para la transformación de lavas a pupas, pueden ser blancos potenciales para el diseño de estrategias de control dirigidas a interrumpir el desarrollo larvario de An. albimanus.

Palabras clave:
expresión génica; neuropéptidos; Anopheles; insecto

Introduction

Insects play prominent roles in supporting human welfare (as food sources and crop pollinators), a number of hematophagous arthropods transmit several infectious diseases.¹1. Organización Mundial de la Salud. Enfermedades transmitidas por vectores 2016. Ginebra: OMS 2016. Available at: http://www.who.int/mediacentre/factsheets/fs387/es/
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Neuropeptides have pleiotropic functions in insects’ physiological processes, including reproduction, development, metabolism, and behavior.⁸8. Nassel DR, Winther AM. Drosophila neuropeptides in regulation of physiology and behavior. Prog Neurobiol 2010;92(1):42-104. https://doi.org/10.1016/j.pneurobio.2010.04.010
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https://doi.org/10.3390/v6114314... Of particular interest are those engaged in mosquito metamorphosis and ecdysis, such as: Crustacean CardioActive Peptide (CCAP), Ecdysis Triggering Hormone (ETH), corazonin and bursicon. CCAP is a cyclic nonapeptide that stimulates heartbeat¹⁰10. Stangier J, Hilbich C, Beyreuther K, Keller R. Unusual cardioactive peptide (CCAP) from pericardial organs of the shore crab Carcinus maenas. Proc Natl Acad Sci U S A 1987;84(2):575-579. https://doi.org/10.1073/pnas.84.2.575
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https://doi.org/10.1002/arch.1045... and bursicon, a heterodimer required for sclerotization, cuticle tanning and wing expansion after eclosion.¹⁶16. Luo CW, Dewey EM, Sudo S, Ewer J, Hsu SY, Honegger HW, et al. Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2. Proc Natl Acad Sci USA 2005;102(8):2820-2825. https://doi.org/10.1073/pnas.0409916102
https://doi.org/10.1073/pnas.0409916102... Prior to ecdysis, corazonin is released into the haemocel and activates the release of pre-ecdysis triggering hormone (PETH) and ETH.¹⁷17. Kim YJ, Spalovska-Valachova I, Cho KH, Zitnanova I, Park Y, Adams ME, et al. Corazonin receptor signaling in ecdysis initiation. Proc Natl Acad Sci USA 2004;101(17):6704-6709. https://doi.org/10.1073/pnas.0305291101
https://doi.org/10.1073/pnas.0305291101... ETH is a peptide that activates neurons that produce the eclosion hormone (EH), CCAP and bursicon.¹⁸18. Kruger E, Mena W, Lahr EC, Johnson EC, Ewer J. Genetic analysis of Eclosion hormone action during Drosophila larval ecdysis. Development 2015;142:4279-4287. https://doi.org/10.1242/dev.126995
https://doi.org/10.1242/dev.126995... Other important neuropeptides involved in growth and development are the insulin like peptides (ILP´s), orcokinins and allatostatins. ILP´s seem to be involved in development, regulation of carbohydrate and lipid metabolism,¹⁹19. Baker KD, Thummel CS. Diabetic larvae and obese flies - emerging studies of metabolism in Drosophila. Cell metabolism 2007;6(4):257-266. https://doi.org/10.1016/j.cmet.2007.09.002
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https://doi.org/10.1016/j.tibs.2007.02.0... and regulation of sleep.²⁴24. Cong X, Wang H, Liu Z, He C, An C, Zhao Z. Regulation of Sleep by Insulin-like Peptide System in Drosophila melanogaster. Sleep 2015;38(7):1075-1083. https://doi.org/10.5665/sleep.4816
https://doi.org/10.5665/sleep.4816... In Aedes aegypti, insulin was shown to stimulate ecdysteroid biosynthesis in the ovaries.²⁵25. Riehle MA, Brown MR. Insulin stimulates ecdysteroid production through a conserved signaling cascade in the mosquito Aedes aegypti. Insect Biochem Mol Biol 1999;29(10):855-860. https://doi.org/10.1016/S0965-1748(99)00084-3
https://doi.org/10.1016/S0965-1748(99)00... Allatostatins, are potential insect growth regulators, which inhibit the production of juvenile hormone (JH);²⁶26. Stay B, Tobe SS, Bendena WG. Allatostatins: Identification, Primary Structures, Functions and Distribution. In: Evans PD, ed. Advances in Insect Physiology. Volume 25. Oxford UK: Oxford University, 1995:267-337. https://doi.org/10.1016/s0065-2806(08)60066-1
https://doi.org/10.1016/s0065-2806(08)60... although it appears to have other functions, including neuromodulation, regulation of muscle contraction, and regulation of enzyme biosynthesis.²⁷27. Bendena WG, Donly BC, Tobe SS. Allatostatins: a growing family of neuropeptides with structural and functional diversity. Ann N Y Acad Sci 1999;897:311-329. https://doi.org/10.1111/j.1749-6632.1999.tb07902.x
https://doi.org/10.1111/j.1749-6632.1999... Orcokinins are involved in the neuronal regulation of ecdysteroidogenesis²⁸28. Yamanaka N, Roller L, Zitňnan D, Satake H, Mizoguchi A, Kataoka H, et al. Bombyx orcokinins are brain-gut peptides involved in the neuronal regulation of ecdysteroidogenesis. J Comp Neurol 2011;519(2):238-246. https://doi.org/10.1002/cne.22517
https://doi.org/10.1002/cne.22517... and circadian locomotor activity²⁹29. Hofer S, Homberg U. Evidence for a role of orcokinin-related peptides in the circadian clock controlling locomotor activity of the cockroach Leucophaea maderae. J Exp Biol 2006;209:2794-2803. https://doi.org/10.1242/jeb.02307
https://doi.org/10.1242/jeb.02307... and the control of vitellogenesis.³⁰30. Ons S, Bellés X, Maestro JL. Orcokinins contribute to the regulation of vitellogenin transcription in the cockroach Blattella germanica. J Insect Physiol 2015;82:129-133. https://doi.org/10.1016/j.jinsphys.2015.10.002
https://doi.org/10.1016/j.jinsphys.2015....

Understanding the expression of these neuropeptides in developmental stages of insect vectors may open opportunities for identifying possible candidate molecules to design species-specific insecticides for biological control.⁹9. O’Neal ST, Samuel GH, Adelman ZN, Myles KM. Mosquito-borne viruses and suppressors of invertebrate antiviral RNA silencing. Viruses 2014;6(11):4314-4331. https://doi.org/10.3390/v6114314
https://doi.org/10.3390/v6114314... In this paper, we examined the expression of eight neuropeptides, three associated to ecdysis, during the ontogeny of the mosquito Anopheles albimanus, one of the main malaria vectors in Mexico and Central America.³¹31. Sinka ME, Rubio-Palis Y, Manguin S, Patil AP, Temperley WH, Gething PW, et al. The dominant Anopheles vectors of human malaria in the Americas: occurrence data, distribution maps and bionomic precis. Parasit Vectors 2010;3:72. https://doi.org/10.1186/1756-3305-3-72
https://doi.org/10.1186/1756-3305-3-72...

Materials and methods

Biological samples of Anopheles albimanus

Larval, pupae and adults mosquitoes were obtained from the insectary of the Center for Research on Infectious Diseases (CISEI), National Institute of Public Health (Mexico). Larvae were grown under controlled temperature conditions (29-33°C) and humidity (70-80%), with a 12h light/12h dark photoperiod. Larvae were fed with grinded cat chow (Whiskas 0.1876 grams every 24 hours) in trays with 1.5 liters of water at 25-30°C at larval density of 200 each tray. Larval samples were taken every 24 h after egg hatching and adult mosquitoes were collected 24 hours after emergence. Because it was difficult to differentiate between larvae instars by morphology, larvae were measured and defined as first instar, larvae of 2 mm, second instar, larvae of 4 mm, third instar, larvae of 5mm and fourth instar, larvae of 6 mm in length. Groups of 8 larvae, 8 pupae and 8 adult mosquitoes were placed in 500 μl of Trizol (Ambion) and stored at -20°C until use.

Total RNA purification

Total RNA was extracted as previously described.³²32. Chomczynski P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 1993;15:532-537. Briefly, samples in Trizol were macerated with a biovortex, 10 pulses per 1 minute with a 30 second break. Samples were centrifuged for 5 min at 8 000 x g to remove the debris, the supernatant was added to 100 µl of chloroform (Sigma-aldrich), mixed and centrifuged for 15 min at 10 000 x g at 4°C, the aqueous phase was recovered and 250 µl of cold isopropanol (Sigma-Aldrich) were added, mixed and incubated at -20 °C for 1 hour. Samples were centrifuged at 10 000 x g for 10 min, the pellets were washed with 500 µl of 75% ethanol and centrifuged at 7 000 x g for 5 min; the supernatants were removed and the pellets were-suspended in 20 µl of DEPC-treated water (diethylpyrocarbonate) (Sigma-aldrich). RNA was quantified with Nanodrop and visualized using electrophoresis in agarose gels stained with ethidium bromide, to confirm integrity.

cDNA Synthesis

One µg of total RNA of each sample was treated with DNAse (ThermoScientific® ™ 200 U/µl). cDNA was synthetized, the reaction included: 250 ng oligodT (Thermo Scientific), 5X RT buffer (Promega), 2.5 mM dideoxynucleotide mixture (dNTP’s) (Promega), 10U/µl RNAse Inhibitor (RNAsin) and 5 U/µl reverse transcriptase enzyme (Promega). All samples were adjusted to a final volume of 40 µl of DEPC-treated water and incubated at 42 °C for 90 minutes. The synthesized cDNAs were stored at -70 °C until use.

Quantitative real time PCR (qPCR)

Specific oligonucleotides corresponding to CCAP, corazonin, ETH, ILP2, ILP5, allatostatin-A, orcokinin, and bursicon genes were designed using the Oligo 3.1 Program Analyzer (http://www.idtdna.com/site), based on the nucleotide sequences identified in the genome (https://www.vectorbase.org/organisms/anopheles-albimanus) and a brain transcriptome of An. albimanus.³³33. Martínez-Barnetche J, Gómez-Barreto RE, Ovilla-Muñoz M, Téllez-Sosa J, García López DE, Dinglasan RR, et al. Transcriptome of the adult female malaria mosquito vector Anopheles albimanus. BMC Genomics 2012;13:207. https://doi.org/10.1186/1471-2164-13-207
https://doi.org/10.1186/1471-2164-13-207... All quantitative RT-PCR assays were performed in duplicate with three biological replicates for each experiment. As an endogenous control, an oligonucleotide corresponding to the actin gene of An. albimanus was used. Real-time PCR assays were performed on an Applied Biosystems (ABI) Step One Plus Real-Time PCR System using the reaction mixture Master Mix (2X) SYBRGreen universal qPCR (ThermoScientific). The PCR program was: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 seconds and 64°C for 1 min, then 95 °C for 15 sec, 64 °C for 15 sec, and 95 °C for 15 sec, for one cycle. The specificity of the SYBR green PCR signal was confirmed by a melting curve analysis and 1.2% agarose gel electrophoresis. The expression of each neuropeptide in different mosquito development stages was compared using the 2-DDCt method³⁴34. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25(4):402-408. https://doi.org/10.1006/meth.2001.1262
https://doi.org/10.1006/meth.2001.1262... and calculated as fold increase comparing the expression of each neuropeptide in each stage against actin and tested with one-way ANOVA followed by the Kruskal-Wallis post-test (α= 0.05). Finally, graphical data were shown as percentage of expression by stage.

Results

The transcription pattern of CCAP, corazonin, ETH, ILP2, ILP5, bursicon, allatostatin-A and orcokinin neuropeptides in larvae from first to fourth instar, pupa black and mosquito was measured.

Neuropeptide expression associated to Ecdysis

The transcription pattern of CCAP, corazonin and ETH transcripts varied among the different mosquito developmental stages. The highest expression of the three neuropeptide transcripts was observed in fourth instar larvae (70.8, 76.5 and 60.2%, respectively) (figure 1). CCAP transcription increased from first to third instar larvae (1.81 to 21.3%) but, in pupae and adult mosquitoes, transcription levels decreased to 4 and 0.3%, respectively (p< 0.001) (figure 1A). The corazonin transcript had similar expression profile, in first to third instar larvae, transcription levels were 4 to 1.6%, respectively but, in pupae and adult mosquitoes, transcription levels decreased to 0.1 and 2.5%, respectively ( < 0.001) (figure 1B). The transcription of ETH increased from first to third instar larvae (0.1 to 13.7%, respectively), in pupae the transcription decreased to 1.5% but in adult mosquitoes, it increased to 14.5% (p< 0.001) (figure 1C). A schematic representation of the neuropeptide variation among mosquito stages is shown in figure 1D.

ILP 2, ILP5, bursicon, allatostatin-A and orcokinin

The transcription of ILP 2 was a high in most stages: 47% (first instar), 45.5% (second instar), 45% (third instar), 64.5% (pupae) and 61.6% (mosquito) (p< 0.001), the lowest transcription level of ILP2 was in fourth instar larvae (1.2%) (figure 2). Allatostatin-A and bursicon transcription occurred in fourth instar larvae. ILP5 expression decreased as the mosquito development progressed: 9, 6.5, 1.4, 0.2 and 0.1% in first, second, third and fourth instar larvae and pupae, respectively; but increased again in adult mosquitoes (6.5%) to similar values to that of fourth instar larvae (p< 0.001) (figure 2). The transcription of bursicon increased from first instar (11.2%) to third instar larvae (34.2%). Its transcription decreased in fourth instar larvae (1.5%), increased again in pupae (27.6%), but in adult mosquitoes the transcript expression was lower (18.3%) (p< 0.001) (figure 2). No detectable transcription variations during the mosquito development were observed for orcokinin and allatostatin-A. The transcription pattern of orcokinin was 15.5, 0.7, 5.3, 2, 0.4, and 0.1% in first, second, third and fourth instar larvae, pupae and adult mosquitoes, respectively. The transcription pattern of allatostatin-A was 2.3, 4.94, 2.9, 1.1, 4.2, and 5.8% in first, second, third and fourth instar larvae, pupae and adult mosquitoes, respectively (figure 2).

Discussion

In this paper we analyzed the transcription pattern of eight neuropeptides, during the development of An. albimanus mosquitoes. Mosquitoes have a well-characterized development process from egg hatching to adults. Neuropeptides are essential during their ontogeny and together with their receptors, have been proposed as candidates for the generation of larvicides that disrupt their development. Currently, a number of peptidomimetics with c-terminal motifs PRXamide³⁵35. Jiang H, Wei Z, Nachman RJ, Kaczmarek K, Zabrocki J, Park Y. Functional characterization of five different PRXamide receptors of the red flour beetle Tribolium castaneum with peptidomimetics and identification of agonists and antagonists. Peptides 2015;68:246-252. https://doi.org/10.1016/j.peptides.2014.11.004
https://doi.org/10.1016/j.peptides.2014.... and FGLamide³⁶36. Xie Y, Zhang L, Zhang C, Wu X, Deng X, Yang X, et al. Synthesis, biological activity, and conformational study of N-methylated allatostatin analogues inhibiting juvenile hormone biosynthesis. J Agric Food Chem 2015;63(11):2870-2876. https://doi.org/10.1021/acs.jafc.5b00882
https://doi.org/10.1021/acs.jafc.5b00882... that confer an increase in their stability and bioavailability have been studied for their use in pest control. For example, a β-amino acid pyrokinin analog (Ac-Y[β3F]TPRLamide) accelerates an irregular pupation in the flesh fly Sarcophaga bullata.³⁷37. Nachman RJ, Wang XJ, Etzkorn FA, Aziz OB, Davidovitch M, Kaczmarek K, et al. Evaluation of a PK/PBAN analog with an (E)-alkene, trans-Pro isostere identifies the Pro orientation for activity in four diverse PK/PBAN bioassays. Peptides 2009;30(7):1254-1259. https://doi.org/10.1016/j.peptides.2009.04.017
https://doi.org/10.1016/j.peptides.2009.... A PK/PBAN antagonist lead (RYF[dF]PRLa) was reported as a potent inhibitor of a sex pheromone biosynthesis in Heliothis peltigera.³⁸38. Nachman RJ, Teal PE, Aziz OB, Davidovitch M, Zubrzak P, Altstein M. An amphiphilic, PK/PBAN analog is a selective pheromonotropic antagonist that penetrates the cuticle of a heliothine insect. Peptides 2009;30(3):616-621. https://doi.org/10.1016/j.peptides.2008.09.024
https://doi.org/10.1016/j.peptides.2008.... Using a diapause hormone (DH) agonistic and/or antagonistic peptidomimetics the pupal diapause of Helicoverpa zea was disrupted³⁹39. Zhang Q, Nachman RJ, Kaczmarek K, Zabrocki J, Denlinger DL. Disruption of insect diapause using agonists and an antagonist of diapause hormone. Proc Natl Acad Sci USA 2011;108(41):16922-16926. https://doi.org/10.1073/pnas.1113863108
https://doi.org/10.1073/pnas.1113863108... and analogs of the pyrokinin, insect kinin and insect tachykinin families showed aphicidal activity, that was better than some commercially available aphicides.⁴⁰40. Smagghe G, Mahdian K, Zubrzak P, Nachman RJ. Antifeedant activity and high mortality in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae) induced by biostable insect kinin analogs. Peptides 2010;31(3):498-505. https://doi.org/10.1016/j.peptides.2009.07.001
https://doi.org/10.1016/j.peptides.2009.... In mosquitos, only CCAP,³3. Estevez-Lao TY, Boyce DS, Honegger HW, Hillyer JF. Cardioacceleratory function of the neurohormone CCAP in the mosquito Anopheles gambiae. J Exp Biol 2013;216:601-613. https://doi.org/10.1242/jeb.077164
https://doi.org/10.1242/jeb.077164... FMRFamide-like peptides (FLP),⁴¹41. Hillyer JF, Estévez-Lao TY, de la Parte LE. Myotropic effects of FMRFamide containing peptides on the heart of the mosquito Anopheles gambiae. Gen Comp Endocrinol 2014;202:15-25. https://doi.org/10.1016/j.ygcen.2014.03.048
https://doi.org/10.1016/j.ygcen.2014.03.... corazonin,⁴²42. Hillyer JF, Estévez-Lao TY, Funkhouser LJ, Aluoch VA. Anopheles gambiae corazonin: gene structure, expression and effect on mosquito heart physiology. Insect Mol Biol 2012;21(3):343-355. https://doi.org/10.1111/j.1365-2583.2012.01140.x
https://doi.org/10.1111/j.1365-2583.2012... bursicon,⁴³43. Honegger HW, Estévez-Lao TY, Hillyer JF. Bursicon-expressing neurons undergo apoptosis after adult ecdysis in the mosquito Anopheles gambiae. J Insect Physiol 2011;57(7):1017-1022. https://doi.org/10.1016/j.jinsphys.2011.04.019
https://doi.org/10.1016/j.jinsphys.2011.... short neuropeptide F (sNPF),⁴⁴44. Garczynski SF, Crim JW, Brown MR. Characterization and expression of the short neuropeptide F receptor in the African malaria mosquito, Anopheles gambiae. Peptides 2007;28(1):109-118. https://doi.org/10.1016/j.peptides.2006.09.019
https://doi.org/10.1016/j.peptides.2006.... AKHI and AKHII⁴⁵45. Kaufmann C, Brown MR. Adipokinetic hormones in the African malaria mosquito, Anopheles gambiae: identification and expression of genes for two peptides and a putative receptor. Insect Biochem Mol Biol 2006;36(6):466-481. https://doi.org/10.1016/j.ibmb.2006.03.009
https://doi.org/10.1016/j.ibmb.2006.03.0... have been studied during the ontogeny of An. gambiae.

This is first report of the expression of neuropeptides associated to several functions during ontogeny of An. albimanus. The high transcription of CCAP, ETH and corazonin in fourth instar larvae suggests that these neuropeptides associated with ecdysis begin their expression in this instar, and that corazonin and CCAP are required to activate ETH and start the ecdysis. Unlike these results, CCAP and corazonin are expressed in different developmental stages of An. gambiae,³¹31. Sinka ME, Rubio-Palis Y, Manguin S, Patil AP, Temperley WH, Gething PW, et al. The dominant Anopheles vectors of human malaria in the Americas: occurrence data, distribution maps and bionomic precis. Parasit Vectors 2010;3:72. https://doi.org/10.1186/1756-3305-3-72
https://doi.org/10.1186/1756-3305-3-72... Specifically, CCAP mRNA levels increased in second instar larvae, decreased in third instar larvae and then increase to maximum levels in callow pupa stage.³3. Estevez-Lao TY, Boyce DS, Honegger HW, Hillyer JF. Cardioacceleratory function of the neurohormone CCAP in the mosquito Anopheles gambiae. J Exp Biol 2013;216:601-613. https://doi.org/10.1242/jeb.077164
https://doi.org/10.1242/jeb.077164... While corazonin showed a similar transcription profile in larvae of An. gambiae and An. albimanus the increase to maximum levels in An. gambiae occurred in young adults.⁴²42. Hillyer JF, Estévez-Lao TY, Funkhouser LJ, Aluoch VA. Anopheles gambiae corazonin: gene structure, expression and effect on mosquito heart physiology. Insect Mol Biol 2012;21(3):343-355. https://doi.org/10.1111/j.1365-2583.2012.01140.x
https://doi.org/10.1111/j.1365-2583.2012... We speculate that the different expression pattern of CCAP, corazonin and ETH during the ontogeny of An. albimanus and An. gambiae is due to factors associated to development, such as: temperature, food and stress; but also to different environmental adaptations of both mosquito species such as resistance to insecticides, colonization of areas with higher altitudes to 1 500 masl. The transcription expression profile of neuropeptides that we report here and other characteristics such as the short peptide sequence (CCAP, nine amino acid residues, corazonin, eleven amino acid residues and ETH, seventeen amino acid residues), the quaternary structure, the potential generation of peptidomimetics and modifications in the amino acids residues to obtain synthetic structures with desired molecular properties (increased biostability, specificity, and permeability) provide advantages to these neuropeptides for further functional studies in An. albimanus.

Orcokinin and ILP5 showed the highest transcription levels in first instar larvae, while ILP2 showed the highest transcription levels in pupae, allatostatin-A in adult mosquitoes and bursicon in third instar. On the other hand, allatostatin-A , bursicon and ILP2 showed lower transcription levels of in fourth instar larvae while orcokinin in adult mosquitoes and ILP5 in pupae. It would be interesting to conduct further functional studies and explore the possibility of blocking the expression of each of these neuropeptides during larval development of mosquito.

Although we explored eight neuropeptides and analyzed only larvae, pupae and 24 hours post-emergence adult stages, it is clear that many of the neuropeptides are expressed in different intensities or are specific to different stages; and it would be interesting to also investigate their expression in eggs and post emergence individuals. We also recognize that other important stages as eggs, brown pupae and adult stages older than 24 hours post emergency require further studies. However, the encouraging results of the present study will hopefully stimulate further studies that will explore neuropeptides as target molecules in larval control of diseases-transmitting insects. Undoubtedly detailed studies are needed to identify specific candidates to fight anophelines mosquitoes, vectors of malaria, a disease that remains a public health problem worldwide.

Acknowledgments

This study was supported by the Consejo Nacional de Ciencia y Tecnología de México (Conacyt) (Grant 243171). Alejandro Alvarado Delgado is a PhD student in Programa de Doctorado en Ciencias Biomédicas UNAM, and appreciates the support given by Conacyt (Beca 98230).

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