Abstract
In Ethiopia, mosquito larva control interventions are given less emphasis due to a number of factors including lack of precise information on potential breeding habitats and complicated vector biology. Hence, the objective of this study was to assess the spatio-temporal variation and habitat characterization of Anopheline larvae in Bure district, Northwestern Ethiopia. Longitudinal larval sampling was done in 3 villages (Bukta, Workimdr and Shnebekuma) for a year (July 2015 - June, 2016). Sampling was done one time per month. Documentation of habitat type and environmental characteristics were done in each round of sampling. A total of 3490 Anophelines larvae were collected in a year. Seven larval species were identified morphologically: Anopheles gambiae s.l, An. funestus s.l, An. pharoensis. An. demeilloni, An. coustani, An. squamosus, An. cinereus. Of these, An. demeilloni (1.97 ± 0.03) and An. gambiae s. l (1.79 ± 0.06) were the predominant larvae, whereas An. pharoensis (0.18 ± 0.04) was the least representative (p < 0.01). Anophelines larvae were found in various breading habitats, but the mean density of Anophelines larvae in dam- edge (0.45 ± 0. 19) was relatively smaller than other habitats (p < 0.01). The large densities of An. gambiae s.l and An. funestus s.l larvae were sampled from natural, permanent and temporary aquatic habitats that had clean and standing water, without mats of algae, vegetation’s and plants. Multiple linear regression analysis showed that only standing water was found to be the main predictor of the density of An. gambiae s.l density (R2 = 0.457; p < 0.016). In conclusion, the larvae of the two most important malaria vectors of Ethiopia, An. gambiae and An. funestus s.l, were collected through the year.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The data sets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.
References
Afrane YA, Zhou G, Lawson BW Githeko AK, Yan G (2006) Effects of micro-climatic changes caused by deforestation on the survivorship and reproductive fitness of Anopheles gambiae in western Kenya highlands. Am J Trop Med Hyg 74:772–778
Animut A, Gebre-Michae T, Balkew M, Lindtjorn B (2012) Abundance and dynamics of Anopheline larvae in a highland malarious area of south-Central Ethiopia. Parasit Vectors 5:1–9. https://doi.org/10.1186/1756-3305-5-117
Asmare Y, Hopkins RJ, Tekie H, Hill SR, Ignell R (2017) Grass pollen affects survival and development of larval Anopheles arabiensis (Diptera: Culicidae). J Insect Sci 17:1–8. https://doi.org/10.1093/jisesa/iex067
Ayele DG, Zewotir TT, Mwambi HG (2012) Prevalence and risk factors of malaria in Ethiopia. Malar J 11:1–8
Carter C (2013) Summary proceedings 4th annual malaria control program review Ethiopia and Nigeria. Carter Center, Atlanta, Georgia., pp1-19
Carter R, Mendis KN, Roberts D (2000) Spatial targeting of interventions against malaria. Bull World Health Organ 78:1401–1411
CDC (2015) Ecology of malaria. CDC-24/7, Atlanta, GA, pp 30329–34027 Available at: https://www.cdc.gov/malaria/about/biology/ecology.html
Chan H, Githeko AK, Zhou G, Githure JI, Yan G (2006) New records of Anopheles arabiensis breeding on the Mount Kenya highlands indicate indigenous malaria transmission. Malar J 5:1–4. https://doi.org/10.1186/1475-2875-5-17
Chase JM, Knight TM (2003) Drought-induced mosquito outbreaks in wetlands. Ecol Lett 6:1017–1024. https://doi.org/10.1046/j.1461-0248.2003.00533.x
Checchi F, Cox J, Balkan S, Tamrat A, Priotto G, Alberti KP, Zurovac D, Guthmann JP (2006) Malaria epidemics and interventions, Kenya, Burundi, southern Sudan, and Ethiopia, 1999–2004. Emerg Infect Dis 12:1477–1484. https://doi.org/10.3201/eid1210.060540
Coosemans M, Wery M, Mouchet J, Carnevale P (1992) Transmission factors in malaria epidemiology and control in Africa. Mem Inst Oswaldo Cruz 87:385–391. https://doi.org/10.1590/s0074-02761992000700065
Dejenie T, Yohannes M, Assmelash T (2011) Characterization of mosquito breeding sites in and in the vicinity of Tigray micro-dams. Ethiop J Health Sci 21:547–564
Dotson EM (2011) Methods in Anopheles research /MR4. CDC, national institution of health and ATCC. CDC Atlanta GA, USA. https://www.beiresources.org/portals/2/MR4/MR4_Publications/Methods%20in%20Anopheles%20Research%202014/2014MethodsinAnophelesResearchManualFullVersionv2tso.pdf
Ebi KL (2009) Managing the changing health risks of climate change. By Elsevier B.V. Curr Opin inf Environ Sustain 1:107–110. https://doi.org/10.1016/j.cosust.2009.07.011
Fillinger U, Lindsay W (2011) Larval source management for malaria control in Africa: myths and reality. Malar J 10(353):1–10
Fillinger U, Sonye G, Killeen GF, Knols BG, Becker N (2004) The practical importance of permanent and semi-permanent habitats for controlling aquatic stages of Anophele gambiae s.l. mosquitoes: operational observations from a rural town in western Kenya. Tropical Med Int Health 9:1274–1289
Fisiha MY (2002) Do local environmental changes resulting from the construction of microdams lead to increased malaria transmission in Tigra, Ethiopia? Dissertation, University of Durham. Durham E-Theses Online: hhttp://etheses.dur.ac.uk/3867/1/3867_1428.pdf?UkUDh:CyT=
Forstinus NO, Ikechukwu NE, Emenike MP, Osita O (2015) Anopheline mosquitoes and the malaria scourge. Int J Mosq Res 2:200–204
Gillies MT, Coetzee M (1987) A supplement to the Anophelinae of Africa south of the Sahara (Afrotropical region). S Afr Inst Med Res 55:1–125
Gimnig JE, Ombok M, Kamau L, Hawley AW (2001) Characteristics of larval Anopheline (diptera: Culicidae) habitats in western Kenya. J Med Entomol 38:282–288. https://doi.org/10.1603/0022-2585-38.2.282
Gimnig JE, Ombok M, Otieno S, Kaufman MG, Vulule JM, Walker ED (2002) Density dependent development of Anopheles gambiae (Diptera: Culicidae larvae in artificial habitats. J Med Entomol 39, 162-72. https://doi.org/10.1603/0022-2585-39.1.162
Gone T, Balkew T, Gebre-Michael T (2014) Comparative entomological study on ecology and behavior of Anopheles mosquitoes in highland and lowland localities of Derashe district, southern Ethiopia. Parasit Vectors 7:1–9. https://doi.org/10.1186/s13071-014-0483-9
Heggenhougen HK, Hackethal V, Vivek P (2003) The behavioral and social aspects of malaria and its control. WHO. TDR/STR/SEB/ 03(1):21–35pp Available at https://www.who.int/tdr/about/seb_malaria.pdf
Imbahale SI, Paaijmans KP, Mukabana WR, Lammeren RV, Giheko AK, Takken W (2011) A longitudinal study on Anopheles mosquito larval abundance in distinct geographical and environmental settings in western Kenya. Malar J 10:1–11
Ishak H, Tawaddud BI, Amiruddin R (2014) Effects of environmental and nutritional factors to the density of larvae Anopheles spp in coastal endemic Bulukumba, Indonesia. Int J Curr Res Acad Rev 2:6–13
Kebede A, Mccann JC, Kiszewski AE, Ye-Ebiyo Y (2005) New evidence of the effects of agro-ecologic change on malaria transmission. Am J Trop Med Hyg 73:676–680. https://doi.org/10.4269/ajtmh.2005.73.676
Kenea O, Balkew M, Gebre-Michael T (2011) Environmental factors associated with larval habitats of anopheline mosquitoes (Diptera: Culicidae) in irrigation and major drainage areas in the middle course of the Rift Valley, Central Ethiopia. J Vector Borne Dis 48:85–92
Kibret S, McCartney M, Lautze J and Jayasinghe NM (2009) Malaria transmission in the vicinity of impounded water: evidence from the Koka reservoir, Ethiopia. Colombo, Sri Lanka: international water management institute (IWMI) research report 132, Colombo Sri Lanka. Available at: www.iwmi.org/Publications/IWMI_Research_Reports/index.aspx
Kibret S, Lautze J, Boelee E, McCartney M (2012) How does an Ethiopian dam increase malaria? Entomological determinants around the Koka reservoir. Tropical Med Int Health 17:1320–1328. https://doi.org/10.1111/j.1365-3156.2012.03077.x
Kibret S, Wilson GG, Ryder D, Tekie H, Petros B (2017) Malaria impact of large dams at different eco-epidemiological settings in Ethiopia. Trop Med Health 45:1–14. https://doi.org/10.1186/s41182-017-0044-y
Kindu M, Aklilu E, Balkew M, Gebre-Michae T (2018) Study on the species composition and ecology of anophelines in Addis Zemen, South Gondar, Ethiopia. Parasit Vectors 11:215. https://doi.org/10.1186/s13071-018-2701-3
Kiszewski A, Mellinger A, Spielman A, Malaney P, Sachs SE, Sachs J (2004) A global index representing the stability of malaria transmission. Am J Trop Med Hyg 70:486–498
Kudom AA (2015) Larval ecology of Anopheles coluzzii in Cape Coast, Ghana: water quality, nature of habitat and implication for larval control. Malar J 14:1–13. https://doi.org/10.1186/s12936-015-0989-4
Kweka EJ, Munga S, Himeidan Y, Githeko AK, Yan G (2015) Assessment of mosquito larval productivity among different land use types for targeted malaria vector control in the western Kenya highlands. Parasit Vectors 8:1–6. https://doi.org/10.1186/s13071-015-0968-1
Li L, Bian L, Yakob L, Zhou U, Yan G (2009) Temporal and spatial stability of Anopheles gambiae larval habitat distribution in western Kenya highlands. Int J Health Geogr 8:1–10
Liu X, Liu Q, Guo Y, Jiang J, Ren D, Zhou G, Zheng C, Liu J, Chen Y, Li HS, Li HZ, Li Q (2012) Random repeated cross-sectional study on breeding site characterization of Anopheles sinensis larvae in distinct villages of Yongcheng city, people republic of China. Parasit Vectors 5(58):1–10 Available at http://www.parasitesandvectors.com/content/5/1/58
Machault V, Gadiaga L, Vignolles C, Jarjaval F, Bouzid S, Sokhna C, Lacaux J, Trape J, Rogier C, Pages F (2009) Highly focused anopheline breeding sites and malaria transmission in Dakar. Malar J 8:1–18. https://doi.org/10.1186/1475-2875-8-138
Majambere S, Fillinger U, Sayer DR, Green C, Lindsay SW (2008) Spatial distribution of mosquito larvae and the potential for targeted larval control in the Gambia. Am J Trop Med Hyg 79:19–27. https://doi.org/10.4269/ajtmh.2008.79.19
Mala AO, Irungu LW, Shililu JI, Muturi EJ, Mbogo CM, Njagi JK, Githure JI (2011) Dry season ecology of Anopheles gambiae complex mosquitoes at larval habitats in two traditionally semi-arid villages in Baringo, Kenya. Parasit Vectors 4:1–10
Manguin S and Boete C (2011) Global impact of mosquito biodiversity, human vector borne diseases and environmental change. In: the importance of biological interactions in the study of biodiversity. Licensee IntechOpen. https://cdn.intechopen.com/pdfs/20136/InTech-Global_impact_of_mosquito_biodiversity_human_vector_borne_diseases_and_environmental_change.pdf
Midekisa A, Senay G, Henebry GM, Semuniguse P, Wimberly MC (2012) Remote sensing-based time series models for malaria early warning in the highlands of Ethiopia. Malar J 11:1–9 Available at: http://openprairie.sdstate.edu/nrm_pubs/25
Midekisa A, Beyene B, Mihretie A, Bayabil E, Wimberly MC (2015) Seasonal associations of climatic drivers and malaria in the highlands of Ethiopia. Parasit Vectors 8:1–11
Miller JR, Huang J, Vulule J, Walker ED (2007) Life on the edge; African malaria mosquito (Anopheles gambiae s.l.) larvae are amphibious. Naturwissenschaften 94:195–199. https://doi.org/10.1007/s00114-006-0178-y
Minakawa N, Muter CM, Githure JH, Beier JC, Yan G (1999) Spatial distribution and habitat characterization of Anopheline mosquito larvae in western Kenya. Am J Trop Med Hyg 61:1010–1016
Minakawa N, Munga S, Atieli F, Mushinzimana E, Zhou G, Githeko AK, Yan G (2005) Spatial distribution of anopheline larval habitats in western Kenyan highlands: effects of land cover types and topography. Am J Trop Med Hyg 73:157–165. https://doi.org/10.4269/ajtmh.2005.73.157
MoH (2012) National Malaria Guidelines. (3rd edt.). Addis Ababa, Ethiopia. Ethiopia Ministry of Health, Addis Ababa, Ethiopia
Mouchet J, Manguin S, Sircoulon K, Laventure S, Faye O, Onapa AW, Carnevale P, Julvez J, Fontenille D (1998) Evolution of malaria in Africa for the past 40- years: impact of climatic and human factors. J Am Mosq Control Assoc 14:121–130
Mulambalah CS, Siamba DN, Ngeiywa MM, Vulule JM (2011) Anopheles species diversity and breeding habitat distribution and the prospect for focused malaria control in the western highlands of Kenya. Int J Trop Med 6:44–51. https://doi.org/10.3923/ijtmed.2011.44.51
Munga S, Minakawa N, Zhou G, Yun G (2005) Association between land and habitat productivity of malaria vectors in western Kenyan highlands. Am J Trop Med Hyg 74:69–75
Munga S, Minakawa N, Zhou G, Githeko AK, Yan G (2007) Survivorship of immature stages of Anopheles gambiae s.l. (Diptera: Culicidae) in natural habitats in western Kenya highlands. J Med Entomol 44:758–764. https://doi.org/10.1093/jmedent/44.5.758
Mwangangi JM, Mbogo CM, Muturi EJ, Nzovu JG, Githure JI, Yan G, Minakawa N, Novak R, Beier JC (2007a) Spatial distribution and habitat characterization of Anopheles larvae along the Kenyan coast. J Vector Borne Dis 44:44–51
Mwangangi JM, Mbogo CM, Muturi EJ, Nzovu JG, Kabiru EW, Githure JI, Novak RJ, Beier JC (2007b) Influence of biological and physicochemical characteristics of larval habitats on the body size of Anopheles gambiae mosquitoes (Diptera: Culicidae) along the Kenyan coast. J Vector Borne Dis 44:122–127
Najera JA, Zaim M (2002) Malaria vector control - decision making criteria and procedures for judicious use of insecticides WHO pesticide evaluation scheme. WHO reference number: WHO/CDS/WHOPES/2002.5 rev.1. https://www.who.int/whopes/resources/who_cds_whopes_2002.5_rev1/en/
O’Connor CT (1967) The distribution of anopheline mosquitoes in Ethiopia. Mosq News 27:42–55
Oaks SC, Mitchell VS, Pearson GW, Carpenter CCJ (eds) (1991) Malaria: obstacles and opportunities. National Academy Press, Washington, D.C., pp 120–128
Ovazza M, Neri P (1955) Vectors of malaria at highland altitudes in the Addis Ababa region, Ethiopia. Bul Soc Path Exot Fil 48:679–686
Overgaard HJ, Tsuda Y, Suwonkerd W, Takagi M (2002) Characteristics of Anopheles minimus (Diptera: Culicidae) larval habitats in northern Thailand. Environ Entomol 31:134–141. https://doi.org/10.1603/0046-225X-31.1.134
Paaijmans KP (2008) Weather, water and malaria mosquito larvae: the impact of meteorological factors on water temperature and larvae of the afro-tropical malaria vector Anopheles gambiae Giles. University Wageningen, Dissertation
Pennas T, Girma S (2012) Essential malaria actions: Ethiopia: case study. USAID and President Malaria Initiative 1–7. https://www.usaid.gov/sites/default/files/documents/1864/Ethiopia%20Malaria%20Operational%20Plan%20FY%202014.pdf
President’s of Malaria Initiative, PMI. (2014) Ethiopia Malaria Operational Plan FY-2014. 1- 18pp
Ramirez JL, Garver LS, Dimopoulos J (2009) Challenges and approaches for mosquito targeted malaria control. Curr Mol Med 9:116–130. https://doi.org/10.2174/156652409787581600
Rozendaal JA (1997) Vector control: methods for use by individuals and communities. WHO, Geneva
Rueda LM (2008) Global diversity of mosquitoes (Insecta: Diptera: Culicidae) in freshwater. Hydrobiologa 595:477–487. https://doi.org/10.1007/978-1-4020-8259-7_48
Sattler MA, Mtasiwa D, Kiam M, Premji Z, Tanner M, Killeen GF, Lengele C (2005) Habitat characterization and spatial distribution of Anopheles species mosquito larvae in dares salaam (Tanzania) during an extended dry period. Malar J 4(4):1–15. https://doi.org/10.1186/1475-2875-4-4
Shililu J, Ghebremeskel T, Seulu F, Mengistu S, Fekadu S, Zerom M, Fekadu H, Zerom M, Ghebregziabiher M, Sintasath D, Bretas G, Mbogo C, Githure J, Brantly E, Novak R, Beier JC (2003) Larva habitat diversity and ecology of anopheline larvae in Eritrea. J Med Entomol 40:921–929
Silver JB (2008) Mosquito ecology: field sampling techniques. (3rd edt). Springer science business media B.V. https://1lib.nl/book/1258249/39b9bf
Sinka ME, Bangs MJ, Manguin S, Coetzee M, Mbogo CM, Hemingway J, Patil AP, Temperley WH, Gething PW, Kabaria CW, Okara RM, Boeckel TV, Godfray HCJ, Harbach RE, Hay SI (2010) The dominant Anopheles vectors of human malaria in Africa, Europe and the Middle East: occurrence data, distribution maps and bionomic precise. Parasit Vectors 3:1–25. https://doi.org/10.1186/1756-3305-3-117
Siraj AS, Santos-Vega M, Bouma MJ, Yadeta D, Carrascal DR, Pascual R (2014) Latitudinal changes in malaria incidence in highlands of Ethiopia and Colombia. Science 343:1154–1158. https://doi.org/10.1126/science.124432s
Sogoba N, Doumbia S, Vounatsou P, Bagayoko MM, Dolo G, Traore SF, Maıga HM, Toure YT, Smith T (2007) Malaria transmission dynamics in Niono, Mali: the effect of the irrigation systems. Acta Trop 101:232–240. https://doi.org/10.1016/j.actatropica.2007.02.005
Stresman GH (2010) Beyond temperature and precipitation: ecological risk factors that modify malaria transmission. Acta Trop 116:167–172. https://doi.org/10.1016/j.actatropica.2010.08.005
Sunahara T, Ishizaka K, Mogi M (2002) Habitat size: a factor determining the opportunity for encounters between mosquito larvae and aquatic predators. J Vector Ecol 27:8–20
Tesfaye S, Belyhun Y, Teklu T, Mengesha T, Petros B (2011) Malaria prevalence pattern observed in the highland fringe of Butajira, southern Ethiopia: a longitudinal study from parasitological and entomological survey. Malar J 10:1–9
Tesi M (2011) Global warming and health: the issue of malaria in eastern Africa’s highlands. Africa initiative. Discussion No 2. 1-15pp. https://www.files.ethz.ch/isn/143053/no2%20rebrand_0.pdf
Tiku MS, Yewhalaw D, Boets P, Ahmed A, Duchateau L, Speybroeck N, Vanwambeke SO, Legesse W, Meester LD, Goethals P (2013) Physicochemical and biological characterization of anopheline mosquito larval habitats (Diptera: Culicidae): implications for malaria control. Parasit Vectors 6:7–14
Toyama Y, Ota M, Molla G, Beyene B (2016) Sharp decline of malaria cases in the Burie Zuria, Dembia, and Mecha districts, Amhara region, Ethiopia, 2012–2014: descriptive analysis of surveillance data. Malar J 15:1–8. https://doi.org/10.1186/s12936-016-1133-9
Tusting LS, Thwing J, Sinclair D, Fillinger U, Gimnig J, Bonner KE, Bottomley C, Lindsay SW (2013) Mosquito larval source management for controlling malaria. Cochrane database of systematic reviews. Issue 8. Art. No.: CD008923. https://doi.org/10.1002/14651858.CD008923.pub2
Varnado WC, Goddard J, Harrison B (2012) Identification guide to adult mosquitoes in Mississippi. Mississippi State University Extension Service, USA
Verrone GA (1962) Outline for the determination of malaria mosquitoes in Ethiopia: part- II- Anopheles larvae. Mosq News 22:364–401
Walker K, Lynch M (2007) Contributions of Anopheles larval control to malaria suppression tropical Africa: review of achievements and potential. Med Vet Entomol 21:2–21. https://doi.org/10.1111/j.1365-2915.2007.00674.x
Washburn JQ (1995) Regulatory factors affecting larval mosquito populations in container and pool habitats: implication for biological control. J Am Mosq Control Assoc 11:279–283 Available at: https://www.biodiversitylibrary.org/content/part/JAMCA/JAMCA_V11_N2_P279-283.Pdf
WHO. (2013) Malaria entomology and vector control: Guide for participants. https://apps.who.int/iris/bitstream/handle/10665/85890/9789241505819_eng.pdf?sequence=1&isAllowed=y
Wimberly MC, Chuang T, Henebry JM, Liu Y, Alemayehu M, Paulos G, Gabriel S (2012) A computer system for forecasting malaria epidemic risk using remotely-sensed environmental data. Managing resources of a limited planet, 6th biennial meeting, Leipzig, Germany. 1-6pp. Available at: https://scholarsarchive.byu.edu/cgi/viewcontent.cgi?article =1685&context=iemssconference
World Health Organization, WHO (2003) Malaria Entomology and Vector Control: Learner’s Guide. (Trial eds.). World Health Organization HIV/AIDS, Tuberculosis and Malaria, Roll Back Malaria. HO/CDS/ CPE/SMT/2002.18. Rev.1: Part I. https://apps.who.int/iris/bitstream/handle/10665/67450/WHO_CDS_CPE_SMT_2002.18_Rev.1_PartI.pdf;sequence=1
Woyessa A (2001) The elucidation of malaria transmission and its prevalence in highland urban area of Akaki town. M.Sc. Thesis, University of Addis Ababa
Woyessa A, Gebre-Micheal T, Ali A (2004) An indigenous malaria transmission in the outskirts of Addis Ababa, Akaki town and its environs. Ethiop J Heal Dev 81:2–7. https://doi.org/10.4314/ejhd.v18i1.9858
Ye-ebiyo Y, Pollack R, Spielman A (2000) Enhanced development in nature of larval Anopheles arabiensis mosquitoes feeding on maize pollen. Am J Trop Med Hyg 63:90–93. https://doi.org/10.4269/ajtmh.2000.63.90·
Ye-ebiyo Y, Pollack R, Kiszewski A, Spielman A (2003) Enhancement of development of larval Anopheles arabiensis by proximity to flowering maize (Zea mays) in turbid water and when crowded. Am J Trop Med Hyg 68:748–752. https://doi.org/10.4269/ajtmh.2003.68.748
Acknowledgements
We thank to Dr. Habtie Teki and Dr. Desta Ejeta for providing chemicals and other facilities to conduct this study. Our gratitude is also extended to Girma Gudisho, my family, and close friends for their unreserved encouragement; Bure district health office experts and people who live in this study area for their collaboration during the entomological data collection.
Funding
We acknowledged Addis Ababa University for partial financial support; Mizan-Tepi University for providing salary until this research work complete, and Malaria Consortium Ethiopia for providing larval sampling equipment’s.
Author information
Authors and Affiliations
Contributions
Tilahun Adugna: designed the study, conducted data collection, laboratory, and data analysis, and, wrote the first draft of the manuscript. Emana Getu and Delenasaw Yewhalaw: designed the study. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interests
The authors declare that they have no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Appendix 1
Appendix 1
Rights and permissions
About this article
Cite this article
Adugna, T., Getu, E. & Yewhalaw, D. Species composition, spatio-temporal variation and habitat characterization of Anopheline larvae in Bure district, northwestern Ethiopia. Int J Trop Insect Sci 41, 2385–2400 (2021). https://doi.org/10.1007/s42690-020-00412-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s42690-020-00412-4