Nig. J. Anim. Prod. 2017, 44(4): 1 - 10 Nigerian Journal of Production Nigerian Society for Animal Production Chromosome numbers, evolutionary relationships and divergence among three breeds of giant african land snails in Nigeria *1 Okon, B., 1 Ibom, L. A., 1 Dauda, A., 1 Bassey, A. E., 2 Awodiran, M. O. and 3 Etukudo, M. O. 1Department of Animal Science, University of Calabar, Calabar, Nigeria 2Department of Zoology, Obafemi Awolowo University, Ile-Ife, Nigeria 3Department of Genetics and Biotechnology, University of Calabar, Calabar, Nigeria *Correspondence: [email protected], GSM +234(0)803 418 3263 Abstract A number of studies have been carried out on the reproductive and growth performance of these breeds of giant African land snails, but not much is documented on chromosome, evolutionary relationships and divergence studies. Forty snails and 10 of each breed of giant African land snails Archachatina marginata (AM), Achatina achatina (AA) and Achatina fulica (AF) and two varieties of A. marginata [A. marginata var. saturalis (AMS) and A. marginata var. ovum (AMO)] were used for the chromosomes numbers analyses. Slides for chromosome identification were prepared using the ovotestes and the cells were examined for spread at metaphase. The haploid (n) chromosome numbers obtained revealed and confirmed that AF,AA, AMS and AMO snails have 27, 30, 28 and 28 chromosomes respectively. Also 13 amino acid sequences were retrieved from the National Centre for Biotechnology Information with accession numbers: ALD09273, AAY62497, ACA 10148 and AKQ 76237 for AM; AKQ 76253, AKQ 76250, CDL 67813, CDL 67813 and AKQ 76249 for AA and SP/P35903, PDB/5CZL, KZM 80032 and YP009049167 for AF snails. The evolutionary history was inferred using the Neighbour-Joining method. Phylogenetic tree analysis by Neighbour- Joining (NJ) was constructed using 10 out of the 13 amino acid sequences. The reliability of the tree was calculated by boostrap confidence value with 1000 bootstrap iterations using MEGA 7 software. The phylogenetic trees showed that these snail breeds are highly divergent. A. achatina was separately related to itself; whereas A. marginata slightly mingled with A. fulica. The degrees of divergence obtained signified that they have evolved from different ancestors. The evolutionary distances were computed with the remaining three amino acid sequences using the Poisson Correction method. Very high and far apart average genetic distances of 0.923±0.018, 0.926±0.018 and 0.926±0.017 were obtained for AA vs AF, AF vs AM and AA vs AM snails respectively, confirming that these snail breeds are genetically far apart between themselves. The results obtained for chromosome numbers, evolutionary relationship and average genetic distances might be a panacea in further selection and molecular classification of giant African land snails (GALS) in Nigeria. Keywords: Chromosome number, Evolutionary distance, Breed, snail, Nigeria

Introduction 2015); as well as in pharmaceutical/cosmetic Nigeria is greatly endowed with large industry in Nigeria (Akpa, 2015). According number of land snails and its domestication to Samet al . (2012), snails are one of the and rearing is recently receiving more commonest sources of animal protein in attention because of its positive impact on Southern Nigeria where the ecology favours agricultural, industrial, pharmaceutical and their continued existence. The meat is high economic transformation agenda (Okon, in protein (12 – 16%) and iron (45 –

1 Divergence among three breeds of giant african land snails in Nigeria

50mg/kg), low in fat (0.05 – 0.08%) and diabetes, hypertension and other heart contains all the amino acids needed for diseases (Onuorah, 2017). The giantAfrican human nutrition (NAERLS, 1995). They land snails (GALS) which belong to the are also rich in vitamins A, E, K and B12 as familyAchatinidae , are made up of four (4) well as essential fatty acids.Also, giant land breeds namely; Archachatina marginata snails are great source of the valuable lectin (Plate 1), Achatina achatina (Plate 2), which possesses anti-cancer properties that Achatina fulica(Plate 3) and Archachatina help in the boosting of immune system. The degneri(Plate 4). But Omole et al. (2000), immune system, according to Onuorah Okon and Ibom (2012) and Okon (2013) (2017) is better equipped to fight against opined that four different popular breeds of cancerous cells. Besides, the low snail found and reared in Nigeria include cholesterol content of snails makes them Archachatina marginata, Achatina suitable meat for those suffering from achatina, Achatina fulica and species.

Plate 1: Archachatina marginata snail Plate 2: Achatina achatina snail

Plate 3: Achatina fulica snail Plate 4: Archachatina degneri snail

2 Okon, Ibom, Dauda, Bassey, Awodiran and Etukudo

Of these species, Smith and Fowler (2003) (2016) opined that the phylogenetic tree is pointed out that onlyAchatina achatina and used to compare the common ancestral Archachatina marginata are “truly giant nucleotide and amino acid sequences of the African land snails” as the largest evolutionary relationships as well as for specimens recorded by [PPQ – Plantt providing information on an evidence of Protection Quarantine] have been in the trans-species evolution which might be range of 750 g and 8 inches shell”. attributed to the coding nature of the Whereas, Adikwu (2012) and Alukoet al . sequence.Alot of research studies have been (2017) noted that the giant African land done and reported on the reproductive and snails consist of only three species namely growth performance of these breeds of giant Achatina achatina, Achatina fulica and African land snails, but not much is Archachatina marginata, probably because documented on chromosome studies, of the smaller size of the Limicolaria evolutionary relationships and divergence. species. Chromosomes are composite Therefore, this study seeks to determine the structures with well-defined structures that chromosome numbers of the two varieties of play vital roles in the process of replication, Archachatina marginata snails, three breeds transcription and regulation of gene of giant African land snails, evolutionary expression. The chromosomes number relationships and their divergence. varies among the different breeds of snails, therefore crossbreeding principles cannot Materials and methods be applied between and among them for Forty snails, 10 of each breed of mature genetic improvement; that is they cannot snailsArchachatina marginata (AM), breed true. Thus, Affonsoet al . (2007) Achatina achatina(AA), Achatina fulica pointed out that the basic information of (AF), and two varieties of Archachatina chromosome number and morphology marginata; A. marginata var. saturalis coupled with simple breeding techniques (AMS) andA. marginata var. ovum (AMO) and light microscopy analysis are useful to were used for the chromosome analysis at explain a variety of subjects ranging from the Obafemi Awolowo University, Ile-Ife, cytotaxonomy to karyotype. Awodiran et Nigeria. The preparation of slides for the al. (2012) reported the haploid (n) identification of chromosome numbers chromosome number of 28, 30, 27 and 24 using the ovotestes were done as described forArchachatina marginata , Achatina by Bassey (2017). The slides were examined achatina, Achatina fulica a n d following the method of Fagbuaroet al . Archachatina papyracea s n a i l s (2002). Cells that were adjudged to contain respectively. Whereas, Fagbuaro et al. well spread metaphase chromosomes were (2002) and Idowu and Akinnusi (2006) had eye karyotyped and then photographed. The earlier observed the diploid (2n) photography was done under oil immersion chromosome number of 56, 44, 54, 28 and using a photo microscope (PWBK5000T, 56 forArchachatina marginata , Achatina PROWAY OPTICS, China). The eye- achatina, Achatina fulica, Limicolaria karyotyping method helped more accurately aurora and Aquatic snails respectively. by identifying homologous chromosome Phylogenetic analysis plays an important pairs as well as differentiate and separate role in the investigation of species diversity artifacts which could easily be confused with as well as novel species identification small chromosomes on the (Surakasiet al. , (2007). Thus, Bibinu et al. photographs/plates.

3 Divergence among three breeds of giant african land snails in Nigeria

A total of 13 amino acid sequences were was drawn to scale, with the branch lengths retrieved from the National Centre for in the same units as those for evolutionary Biotechnology Information with accession distances. The evolutionary distances were numbers: ALD09273, AAY62497, ACA computed using the Poisson correction 10148 and AKQ 76237 for Archachatina method (Zuckerukandl and Pauling, 1965) marginata, AKQ76253, AKQ76250, and are in the units of the number of amino CDL67813, CDL67813 andAKQ76249 for acid substitution per site. All positions Achatina achatina and SP/P35903, containing gaps and missing data were PDB/5CZL, KZM80032 and eliminated. There were a total of 170 YPO09049167 forAchatina fulica snail. positions for the phylogenetic tree and 240 The evolutionary history was inferred using positions for the genetic distances in the final the Neighboring-Joining method (Saitou data set. The evolutionary analyses were and Nei, 1987). Ten amino acid sequences conducted with MEGA7 software (Kumar et were re-selected from the above 13 al., 2016). sequences for the optimal tree with the sum of branch length equals 10.51798688 as Results and discussion shown; while the remaining three amino The number of cells spread examined, the acid sequences were used for the estimation modal haploid and diploid chromosome of evolutionary divergence. The percentage numbers obtained for A. fulica, A. achatina replicate trees in which the associated taxa andA. marginata snailsa re shown in Table 1. clustered in the bootstrap test (1000 The number of spreads used for the replicates) are shown next to the branches chromosome studies of the three breeds and (Felsentein, 1985). The phylogenetic tree two varieties were large and ranged between 180 and 205.

Table 1: The number of metaphase cells, the modal haploid and diploid chromosome numbers of A. fulica, A. achatina, A. marginata var. Ovum and A. marginata var. Saturalis Breed/Variety Number of Modal haploid Modal Diploid spread Number Number A. fulica 205 27 54 A. achatina 200 30 60 A. marginata var. Ovum 180 28 56 A. marginata var. Saturalis 200 28 56 The representative spreads for each breed constant and consistently uniform in the and variety are shown on Plate 5 (A. fulica morphology of chromosome structures chromosomes), Plate 6 (A. achatina studied, and is worthy of note in the study of chromosomes), Plate 7 ( A. marginata var. chromosome numbers. Chromosomes of A. saturalischromosomes) and Plate 8 ( A. marginata(Plates 7 and 8) and A. achatina marginata var. ovum chromosomes). The (Plate 6) snails were large and made up of number of observed metaphase cells were metacentric and acrocentric types.

4 Okon, Ibom, Dauda, Bassey, Awodiran and Etukudo

Plate 5: A. fulica chromosomes Plate 6: A. achatina chromosomes

Plate 7: A.marginata var. Saturalis chromosomes Plate 8: A.marginata var. Ovum chromosomes

Hence, supporting Awodiranet al . (2012) (2012) and Aworidanet al. (2012). Whereas views that most of the chromosomes of A. the haploid (n) chromosome number marginataand A. achatina were large, obtained in this study forA. achatina (n = 30) maximally contracted and were made of was quite higher than the n = 22 reported by metacentrics and acrocentrics while those Fagbuaroet al. (2002) and Idowu and ofA. fulica were smaller and more of Akinnusi (2006); but similar to Choudhury acrocentric types. The diploid (2n) and Mohapatra (1991) and Aworidan et al. chromosome numbers obtained for the (2012) result of n = 30. The disparity snails studied wereA. fulica (2n = 54), A. observed in the haploid chromosome achatina(2n = 60), A. marginata var. number might be attributed to variations in saturalis(2n = 56) and A. marginata var. mitotic index and presence of large number ovum (2n = 56) (Table 1). The haploid of very small chromosomes (Choudhury et chromosome numbers obtained for A. fulica al., 1992), as well as types and sizes of (n = 27) andA. marginata (n = 28) snails chromosomes observed/seen. corresponded to and confirmed the same The Neighbour-Joining trees were haploid chromosome numbers by Idowu constructed to compare the common and Akinnusi (2006), Okon and Ibom ancestral nucleotide and amino acid

5 Divergence among three breeds of giant african land snails in Nigeria sequences of the giant African land snails, obtained by morphological comparisons. since the trees may give useful information Thus, Brown (2002) opined that this for proper understanding of the assumption is often correct. Such evolutionary relationships. The information emanating from this study phylogenetic tree (Fig. 1) obtained revealed would be relevant in further selection of that giant African land snails are highly giant African land snails especially for divergent among the breeds.A. achatina is breeding to improve genetically giant land related to itself because sequences from the snails in Nigeria. same breed tend to cluster together than The results of estimates of evolutionary sequences from other breeds (Fig. 1). divergence between sequences of these snail However,A. marginata slightly breeds (A. fulica, A. achatina and A. intermingled withA. fulica . The degree of marginata) are presented in Table 2. The divergence among these breeds signified upper diagonal represents standard error that the breeds might have evolved from estimate(s); while the lower diagonal is the different ancestors (Dangreet al. , 2009). average genetic distance between breeds This is evidence of trans-species evolution which is also known as the average which might be attributed to the coding nucleotide substitutions per site (Dxy). The nature of the sequence (Bibinuet al ., 2016). Dxy values recorded forA. fulica versus (vs) The assumption is that the gene tree, based A. achatinawas 0.922±0.018, that of A. on molecular data with all its advantages, is fulicavs A. marginata was 0.926±0.018 and more accurate and less ambiguous that ofA . achatina vs A. marginata was representation of the species tree than that 0.926±0.017 (Table 2).

100 AKQ76253 Achatina achatina 9 AKQ76250 Achatina achatina

2 AKD0927 Archachatina marginata SP/P35903 Achatina fulica 2 AAY 62497 Archachatina marginata

ACA10148 Archachatina marginata

CDL 67813 Achatina achatina CDL 67813 Achatina achatina (2) 18 Pdb/5CZL Achatina fulica 33 KZM 80032 Achatina fulica

0.20 Fig. 1: Evolutionary relationships of giant African land snails 6 Okon, Ibom, Dauda, Bassey, Awodiran and Etukudo Table 2: Estimates of evolutionary divergence between sequences Breed Achatina fulica Achatina achatina Archachatina marginata

Achatina fulica 1.000 0.018 0.018 Achatina achatina 0.922 1.000 0.017 Archachatina marginata 0.926 0.926 1.000

These average genetic distances obtained in same breed tend to cluster together than this study were very high. This means that sequences from other breeds; whereas A. these breeds of snails are genetically far marginataslightly intermingled with A. apart between themselves, because the fulica. The degrees of divergence among the average genetic distance (Dxy) is an index snail breeds signified that they might have of divergence between and among the evolved from different ancestors. Besides, breeds. Thus, Vincentet al. (2014) opined the average genetic distances obtained from that the higher the value of Dxy, the far the three breeds of giant African land snails apart the breeds are, which by implication, are very high and far apart, confirming that higher values have lesser orthology and the snail breeds are genetically far apart more paralog and vice versa. Similarly, between themselves. The results obtained on Kanget al . (2008) noted that the larger the chromosome numbers, evolutionary Dxy value, the greater the genetic distance; relationships and average genetic distances while the smaller the Dxy value, the closer might be a panacea in further selection and the genetic distance between the breeds. On classification of giant African land snails the other hand, Mariniet al . (2010) (GALS) in Nigeria. observed ortholog divergence from common ancestors, their different References evolutionary trajectories led to divergence Adikwu, M. U. 2012. Snail production for in the selective constraints on homologous sustainable development and good sites. The results of average genetic health.Proceedings of First distances obtained here for these breeds of International Conference on Giant snails revealed that they might have African Land Snails (GALS), 12th – evolved from different ancestors. Hence, 15th February, 2012. Pg. 3 – 7. these results may also be useful in further Affonso, P. R. A. M., Miranda, V. S., molecular classification. Medrado, A. S., Jacobina, U. P., Bitencourt, J. A., Almeida. J. S. Conclusion and Carneiro, P. L. S. 2007. The results of this study revealed and Chromosomes in focus: basic confirmed that the haploid (n) chromosome cytogenetics, light microcopy and numbers of A. fulica, A. achatina, A. the case of Neotropical fish. In: marginata var. saturalisand A. marginata Modern Research and Educational var. ovum snails are 27, 30, 28and 28 Topics in Microscopy. (Mendez- respectively. The phylogenetic tree showed Vilas, A. and Diaz, J. eds). that these breeds of snails are highly FORMATEX, Bania, Brazil.Pg. 370 divergent.A . achatina was separately – 377. related to itself because sequences from the Akpa, U. A. 2015.Snail farming and

7 Divergence among three breeds of giant african land snails in Nigeria

pharmaceutical/cosmetic industry (Bowdich) (: in Nigeria.Proceedings of 4th : Gastropa). International Caryologin, 44:201 – 208. Conference/workshop on Giant Choudhury, R. C., Pandit, R. K. and African Land Snails(NetGALS), 1st Sahu, T. 1992. Chromosomes of a – 4th June, 2015.Pg. xix –xxviii freshwater gastropod Aluko, F. A., Adesina, E. A., Akanji, A. Lymnaeacuteola Lamarck M., Ogungbesan, A. and (Hymnaeidae: Basommatophora). Fapojuwo, O. E. 2017. Cytologia, 57:143 – 147. Assessment of phenotypic traits of Dangre, D. M., Rathod, D. P., Gade, A. K. Achatinafulicasnails in the derived and Rai, M. K. 2009. An Insilico Savanah Zone of Ogun State, Molecular evolutionary analysis of Nigeria.Journal of Molluscan selected species of Phoma: A Research, 3:27 – 34. comparative approach. Journal Awodiran, M. O., Awepetu, J. I. and Proteomics and Akintoye, M. A. 2012. Study of Bioinformatics.2:295 – 309 . cytogenetic of four species of land Fagbuaro, O., Mosuro, A. A., Bakare, A. snails of the familyAchatinidae in A. and Odaibo, A. B. 2002. South-Western Nigeria, Cytological studies of Ife.Journal of Science, 14(2):233 – Archachatinamarginata a n d 235. Achatinaspecies from South Bassey, A. E. 2017. Phenotypic traits Western Nigeria. Trop ical differentiation, Identification of Veterinary, 20(2):74 – 79. chromosome numbers and Body Felsentein, J. 1985. Confidence limits on weights prediction among three phylogenics: An approach using the breeds and two varieties of bootstrap.Evolution, 39:783 – 791. Archachatinamarginatasnails. Idowu, A. B. and Akinnusi, O. 2006.The M.Sc. Thesis. Department of structure of the ovotestis of the Animal Science, University of common African land snails found Calabar, Calabar, Nigeria.Pg. 118. in Abeokuta, South/West Bibinu, B. S., Yakubu, B. S., Ugbo, S. B. Nigeria.Journal of Animal and Dim, N. I. Production, 33(2):286 – 293. 2016.Computational molecular Kang, J. F.,Li, X. L., Zhou, R.Y.,Li, L. H., analysis of the sequences of BMP Feng., F. J. and Guo, X. L. 2008. 15 gene of ruminants and non- Bioinformatics analysis of ruminants.Open Journal of lactoferrin gene for several Genetics, 6:39 – 40. species.Biochemical Genetics , Brown, T.A. 2002. How genomes replicate 46:312 – 322. and evolve: The reconstruction of Kumar, S., Stecher, G. and Tamura, K. DNA-based phylogenetic trees. 2016. MEGA 7: Molecular Genomes, 16:483 – 505. Evolutionary Genetics Analysis Choudhury, R. C. and Mohapatra, I. version 7.0 for bigger datasets. 1991. Chromosomes of a Molecular Biology and Evolution, pestiferous , Achatina 33:1870 – 1874. (Lissachatina, Fulicafulica Marini, N. J., Thomas, P. D. and Rine, J.(2010. The use of orthologus 8 Okon, Ibom, Dauda, Bassey, Awodiran and Etukudo

sequences to predict the impact of Onuorah, E. 2017. Socio-cultural values of amino acid substitutions on protein giant land snails in Nigeria. function.PLOS Genet .6, el000968. Proceedings of 6t h Annual NAERLS 1995. National Agricultural International Conference/Workshop Extension and Research Liaison of Research Network on Giant Services(FMA & RD, ABU); Snail African Land Snails (NETGALS). production techniques in 4th – 7 th June, 2017, Akure, Nigeria, Nigeria.Extension bulletin No. 108 . Pp. 4 – 5. Forestry series No. 12. Saitou, N. andNei, M. 1987. The Okon, B. 2013. Genetic Improvement of Neighboring-Joining method:Anew Giant African Land Snail method for restructuring (GALS). Proceedings of phylogentic trees. Molecular n d 2 International Biology and Evolution4:406 – 425. Conference/Workshop on Giant Sam, I. M., Okon, B., Ukpanah, U. A. and African Land Snails Edem, W. M. 2017. Body weight (GALS).NetGALS.Abeokuta, prediction of Black and White Nigeria 2nd – 5 th June, 2013, Pg. xii – s k i n n e d xxi. Archachatinamarginatasnails from Okon, B. I. 2015. The place of snail farming Quantitative Traits measurements. in the Agricultural, Industrial, European Journal of Advanced Pharmaceutical and Economic Research in Biological and Life Transformation in Nigeria. Science. 5(1):29 – 35. Keynote Address presented at the Smith, J. W. and Fowler, G. 2003. Pathway 4t h International risk assessment forAchatinide with Conference/Workshop of Research emphasis on the giant African land Network on Giant African Land snails,Achatina fulica (Bowdich) Snails (NetGALS), 1st June, 2015, andLimicolaria aurora (Jay) from Pg. vii – xviii the Caribbean and Brazil, with Okon, B.and Ibom, L. A. 2012. Snail comments on related taxa Breeding and Snailery Achatinaachatina(Linne) and M a n a g e m e n t . F r e s h d e w Archachatinamarginata(swainson) Productions, Calabar, Nigeria Pg. intercepted by PPZ:USDA-APHIS 90. Centre for Plant Health Science and Omole, A. J., Tewe, O.O., Ogundola, F. I., Technology (Internal Report), Oluokun, J. A., Adebowale, E. A., Raleigh, NC, 65. Adetoro, F.O., Olupona, J.A. and Surakasi, P. V.,Wani, A. A., Shouche, Y. S. Fapohunda, J. B. 2000. and Ranade, D. R. 2007. Performance and carcass analysis Phylogenetic analysis of of four popular breeds of snail methanogenic enrichment from commonly reared in South West Lonar Lake in India: Isolation of Nigeria. Proceedings of the 25th Methanocalculussp a n d Annual Conference of Nigeria MethanocalleusSp. Micro.Ecol ., Society for Animal Production 54:697 – 704. (NSAP), March 19th – 23 rd , 2000. Vincent, S. T., Momoh, O. M. andYakubu, Umudike, Nigeria. Pg. 316 – 318. A. 2014. Bioinformatics analysis of

9 Divergence among three breeds of giant african land snails in Nigeria

Beta caseine gene in some selected mammalian species. Research opinions in Animal and Veterinary Sciences, 4:564 – 570. Zuckerkandl, E. and Pauling, L. 1965. Evolutionary divergence and convergence in proteins.Edited in Evolving Genes and Proteins by V. Bryson and H. J. Vogel, Academic Press, NewYork, Pg. 97 – 166.

Received: 11th July, 2017 Accepted: 12th December, 2017

10