The Biology of the African Bonytongue Heterotis niloticus (Cuvier, 1829) from the Lower Niger River at Agenebode in Edo State, Nigeria

Background. 'e African bonytongue, Heterotis niloticus, is readily accommodated in the fresh water of the Niger River. It is available all year round with a large population of juveniles and adults due to its fast growth and versatile feeding habits. 'is commercial fish is a highly preferred source of food because of its high protein content and hardy flesh, thus forming a very important component in the diet of many Nigerians. It is highly valued because of its socioeconomic importance and benefits. Hence, this research is designed with the aim of studying the biology of the African bonytongue, Heterotis niloticus, and providing viable information about its importance in fish culture in order to make an available added variety of culturable and affordable fish species in Nigeria. Results. 'e oesophagus is a muscular organ that is short and distensible. 'e oesophagus leads to the gizzard-like stomach, a reddish bilobed organ that is muscular and tough. H. niloticus is an omnivorous macrophage detritor, consuming a wide variety of bottom-dwelling food items. 'e histology of the gastrointestinal tract reveals four conspicuous layers from the inside to the outside: mucosa, submucosa, inner circular layer and outer longitudinal layer of muscularis, and serosa. 'e presence of numerous mucus glands and longitudinal folds with a prominent columnar epithelium provides durable length or an extension of the gut mucus to aid lubrication and easy passage of food materials, protecting the mucosal epithelium frommechanical or chemical injuries arising from interactions with digestive tract contents or enzymes. In addition, the presence of absorptive cells helps in the absorption of valuable nutritive substances. Conclusions. 'e anatomy of the mouth and gut and the aforementioned histology are modified to accommodate the feeding habits. H. niloticus has a single ovary that rests on the right side of the fish; it is reproductively active as the flood plains rise and peaks at the peak of the rains, being a moderately fecund fish. 'e high fecundity is complemented with peaks of GSI observed during the months of September, October, and November, which reveals spawning periods. Hence, the aforementioned attributes of H. niloticus make the fish a viable fish species for culture.


Background
e African bonytongue, Heterotis niloticus, is a large fish that is widespread in many tropical rivers and freshwater lakes of western and central parts of Africa [1]. In Nigeria, it is distributed along with many freshwater bodies. is fish species is readily accommodated in the fresh water of the Niger River with a large population of juveniles and adults.
is successful adaptation is attributed to abundant varieties of feed and food in the river that is complemented with a versatile mode of feeding which is primarily correlated to the type of alimentary canal and digestive processes [2][3][4]. Heterotis niloticus is an important fish species to the fisherfolk and residents along the Niger River. It is the only species in the genus Heterotis and family Osteoglossidae.
is commercial fish is a highly preferred source of food because of its high protein content and hardy flesh, thus forming a very important component in the diet of many Nigerians. It is highly valued because of its socioeconomic importance and benefits. It has a high growth rate and is available all year round in large quantities and large sizes [5]. Recently, a decline was reported by [3,6]. It is readily preferred and sold smoked or dried.
Fish is an important source of nutrients and also serves as the main source of protein in many Nigerian families. It is also highly recommended by WHO [7] and WHO [8] for the growing and ageing population because of its less tough fibre and immediate digestibility as well as its richness in omega-3 fatty acids needed for good health and brain development in children.
e current fishery (culture, production, and management policies) cannot however satisfy the ever-demanding population, hence the need for optimal production and propagation. H. niloticus is one of the promising species that can be used for culture. To achieve this maximally, a thorough study to obtain knowledge of the fish must be carried out. Hence, this research is aimed at studying the biology of the African bonytongue H. niloticus.

Study Area and Description.
e Niger River is the longest river in West Africa discharging into the Atlantic Ocean in Nigeria. It rises up to 240 km and runs for 4180 km. e river is known to harbor 36 families of freshwater fish and nearly 250 species of which 20 are found nowhere else on earth but Nigeria. From Lokoja, the lower part of the Niger River, the river runs through Agenebode-Ida to Forcados in Delta and Nun River in Rivers State and further. e river is clean and relatively clear. e Niger River floods yearly beginning from September and peaks in November to January. Agenebode is located at latitude 7.10512 and longitude 6.69381 and stretches through an area of 1133 km 2 ( Figure 1). Agenebode waterfront is a very busy part of the Niger located in Edo State, Nigeria, serving water for domestic and industrial uses. Active fishing activities take place along the river for subsistence and commercial fishing.

Collection of Specimens and Sampling.
One hundred and twenty-five (125) fish specimens were collected fortnightly for 12 months from November 2019 to October 2020 from three stations (based on the landing sites of fishing localities) from catch landings of fisherfolk by the use of canoes, gill nets, cast nets, drag nets, fish traps, and calabashes from the Lower Niger River at Agenebode. Fishes were transported to the Laboratory of the Department of Biological Sciences, Edo State University, Uzairue, Edo State. Fishes were rinsed; wiped; identified to species level using guides, keys, and pictures provided by [9][10][11]; and then preserved in 5% formalin.

Morphological Parameters.
e standard length (cm), total length (cm), head length (cm), gape mouth (cm), girth, and weight (g) of H. niloticus were obtained using a graduated ruler and tape, a measuring board, and a digital electronic scale (Sartorius 177). ese were recorded and analyzed. Fin counts and measurements were also taken and recorded. eir content was rinsed off in gently flowing tap water and thereafter fixed in 10% formaldehyde. e samples were then dehydrated through a standard ethanol series to 100%, cleared in xylene, embedded in paraffin wax, then sectioned with a rotatory microtome set at 5-6 pm, deparaffinized, and stained with hematoxylin and eosin. Prepared slides of the gastrointestinal tract were mounted and examined with Olympus Electronic Microscope (model Bino Cxi IS 4381) (PL120) to view and capture features of biological interest.

Anatomy and
Pictures of the mouth, jaws, and pharyngeal teeth were taken with a digital Infinix camera (Model X650B).

Determination of Sex.
Sex could not be obtained from external observation. e stomach was split open from the lower abdominal region to determine the sex. Sex was determined by visual and microscopic observation of the gonads. A fish with one ovary was referred to as female, while a fish with no ovary was referred to as male.

Length-Weight Relationship (LWR).
e total length (TL) and body weight (W) were measured in the fresh samples to the nearest 0.1 cm and 0.01 g, respectively. e length-weight relationships were estimated from the formula W � a L b, where W is the total body weight (g), L is the total length (cm), and a and b are the coefficients of the functional regression between W and L. is relationship was transformed into a linear form by the following equation: (1)

Condition
Factor. e condition factor K was calculated by the following formula given by [12] (K � 100W) where W is the total weight of fish in grams, K is the condition factor, and L is the total length of fish in centimeters. It was calculated for each sex separately and then for the combined sexes. Variations of K with season and size groups were also determined.

Gonadosomatic Index (GSI).
e gonadosomatic index of samples of H. niloticus was determined according to Ugwumba et al. [13] as follows: e monthly catches were sorted by sex. e mature female ovaries were analyzed and used to determine the GSI. International Journal of Zoology Fecundity: the ovary of the mature fish was removed and placed gently in boiling water and allowed to boil for 20 minutes. e eggs became hard enough for easy counting. e boiled eggs were then stored in 5% formalin. 1.00 g of the whole ovary was cut off. e cut section of the eggs was carefully counted and multiplied by the total weight of the ovary to give the total number of eggs. e maturity stages of the ovaries were classified according to Nikolsky [14]: Stage 1, immature; Stage II, quiescent; Stage III, maturing; Stage IV, mature; Stage V, running; Stage VI, spent.
Egg diameter: egg diameter (mm) was measured with an ocular micrometer. A stage micrometer was earlier used to calibrate the microscope. Diameters of twenty eggs randomly selected from each ovary were measured, and their mean was taken as the average egg diameter.

Determination of Food and Feeding Habits.
Each stomach was split open, and the contents were emptied into a Petri dish. e contents were observed under a hand lens, and the food materials were identified.
Frequency of occurrence method (FO): in the frequency of occurrence method, the individual food matter in the stomach was sorted and identified. e number of stomachs in which each food item occurred was expressed as a percentage of the total number of stomachs with food examined.
Number of occurrence method (NO): this method involves counting the number of each food item present in the stomach of a fish and summing these numbers to obtain the total number of all food items found in the stomach. e number of each food item is then expressed as a percentage of the total number of all food items. It was expressed as  International Journal of Zoology percentage number of a food item � total number of a particular food item total number of all food items × 100.
Index of relative importance (IRI): the importance of various food items was determined with the index of food importance following the method of Ugwumba and Ugwumba [15]: where IRI is the index of relative importance, Cn is the percentage of numerical composition, Cw is the percentage of gravimetric composition, and F is the percentage of frequency of occurrence. e dietary compositions for the species examined were expressed as percentages; that is, A food item with %IRI ≥3 is regarded as primary, ≥0.1 to <3 secondary, and ≤0.1 incidental. e relative gut length (RGL): this was expressed as the ratio of the total length of the gut to the total body length [16].
RGL � total length of gut total length of fish , where fish can be classified as herbivorous (RLG >1), carnivorous (RLG <1), or omnivorous (RLG �, or>, or <1). Prey-predator relationship: the relationship between the total body length and the total weight of Heterotis niloticus and prey body weight was determined and described by the following equation [17]: where YL is the prey's body weight (g) and L is the predator's body length (cm) or body weight (g).

Statistical Analyses.
e data obtained from this study was used to derive the means and standard deviation. Linear equations were also used to derive linear regression in the Microsoft Excel 2010 statistical package. Other statistical formulae were given and applied using Microsoft Excel 2010 for analyses.

Results and Discussion
Heterotis niloticus is bronze in color, elongate, compressiform, and covered with strong large cycloid scales. e retractable mouth is terminal with thick lips. In this study, the largest fish species obtained was 64 cm long with a weight of 2787 g ( Figure 2). e dorsal fins are situated towards the posterior of the fish with 33-38 rays, the pectoral fins are paired with no spine and 12 rays, the pelvic fins are paired with no spine and 6 rays, and the anal fin consists of 35 rays. e African bonytongue has 5 rows of scales above the lateral line and 6 below the lateral line, in addition to 48-52 scales on the lateral line. e formula of fin count is thus D33-38: P12: V6: A35-38: C13-15 (48 − 52(6/8)). ese meristic characteristics, number and shape of fins, are often used in the identification of this fish species. e fin count obtained in this study corresponds with other identifications by [10,18]. e mouth opens up to 8% of the total body length making room for numerous and large number of small food materials to be taken in at once (Figures 2 and 3). Its nuchal region is humped; its depth is 3.5-5.0 times its height; its head is short, 3.3% of the total length; the gill openings are covered by a bony operculum that ends with a fleshy attachment, one visible lateral line; and the eyes are sunken and golden brown. e composite anatomy of the interior of the mouth is cartilaginous; this is probably because of its kind of feeding specifics or preference for bone food species or cartilaginous ones, organisms with some form of a skeleton. In the mouth, the tongue is very thick, sort of cartilaginous; this hard nature of the tongue might be an added reason for its name (bonytongue). Behind the tongue is the pharyngeal palate with pebble-like teeth; these palates with teeth are on the roof and floor of the mouth. e sides of the mouth overlap to give room for extensions. e tongue is very thick and cartilaginous and has teeth on the posterior end.
ere are 5 gills on each side. ese gills are folded to extend to the roof of the mouth (Figures 4 and 5). e third has forked cartilage attached to it that extends to the pharyngeal palates. Each gill has a very thick cartilaginous arc, long and slender rakers (178-206), and pairs of filaments (82-86 pairs) which are perfect for sieving tiny food particles such as detritus. Attached to the gills are a pair of the bulblike sacs that are made of an internal anal filled with yellow matter suspected to be used for balancing or as an olfactory organ ( Figure 5). e teeth on the jaws are few and not prominent. Cardiform teeth are present on the premaxilla and dentine and are premandibular. e oesophagus is a muscular organ that is short and distensible.
e oesophagus leads to the gizzard-like stomach, a reddish bilobed organ that is muscular and tough ( Figure 6). is species has frequently been characterized either as an omnivore, insectivore, or detritor, with the latter being, in part, because of its benthic feeding habitats and possession of a gizzard (thick-walled pyloric stomach). is organ is often filled with fluid and not seeds or stones as in the case of birds, and this is also in conformity with the report of [19]. e pyloric ceca are from the end of the gizzard, consisting of two long tubular extensions (17% of the total length of the fish). e function of the pyloric ceca of fish has been postulated as an organ "to store up the food," "putrefy it up," and "concoct it" that is (storage, fermentation, and digestion). Buddington and Diamond [20] reported that the trout, cod, largemouth bass, and striped have pyloric ceca which assist in the digestion of food materials. Further studies showed the presence of enzymes, and the ceca proved to be a major site of sugar, amino acid, and dipeptide uptake, contributing more uptake than the entire remaining alimentary tract in trout and codfishes. e presence of carbohydrates has been reported in the stomach and pyloric ceca of the African bonytongue; this is an indication that ample digestion of carbohydrate materials takes place at this organ site [21]. e intestine also arises from the gizzard and can be clearly distinguished into two sections: the small intestine and large intestine; this organ is very long measuring up to 119% of the whole fish. e intestine ends with the cloaca (Figure 7). is gut length is an indication of herbivorous or omnivorous species. Fishes with long intestines that are more than 100% of their length is often either herbivorous or omnivorous.
e length of the intestine gives room for digestion of the food materials ingested.  Four major and distinct layers make up the gut of H. niloticus which is typical of higher vertebrate animals. e oesophagus composed of four conspicuous layers from the inside to the outside: mucosa, submucosa, inner circular layer and outer longitudinal layer of muscularis, and serosa (Figure 8(a)). e mucosa showed large numbers of the esophageal folds of the lumen of the oesophagus giving ovalshaped appearance in all sections. ese oval folds were thick and elongated and had few gastric glands. e serosa is distinct, and these longitudinal folds may lead to an increase in the capacity of the organ for distension during food transportation [22]. e stomach of H. niloticus (Figure 8(b)) showed dense regions of the gastric gland revealing gastric cells within stratified epithelial cells. e mucosa of the stomach consists of surface and gastric epithelium. e surface epithelium is made up of a single layer of columnar epithelial cells, and the gastric epithelium consists of gastric glands. e lamina propria, in the form of a connective tissue network, lies between the gastric glands.
e muscularis consists of an outer longitudinal and an inner thick circular muscle layer. e columnar epithelium was filled with lamina propria and mucus-secreting cells. e mucous secreted by these cells may be attributed to the lack of salivary glands in fishes; however, reports revealed that mucin is secreted by the esophageal epithelium which is probably a compensation [23,24]. Furthermore, the mucin and mucus secretions are important for the formation of a continuous sheet along the entire wall of the oesophagus, lubrication of food particles, ionic absorption, and protection of the esophageal mucosa against mechanical damage and bacterial invasion, while mucous secretions participate in enzymatic digestion of the ingested food and facilitate its transformation into chime [25,26]. e pyloric ceca (Figure 8(c)) had a dense muscularis, the mucosa had flat epithelium filled with cells, and gastric pits and mucus glands were not visible. e absence of glands and pits with visible macrovilli in the pyloric ceca reveals that this organ is not essential for digestion. Its location between the stomach and the intestine could be primarily to store food and slow down the passage of food into the intestine, providing enzymes and digestive abilities for food. Carnivorous fish species are known to have short intestine, hence the presence of other organs to assist in digestion. e stomach is composed of numerous longitudinal folds, gastric glands, gastric pits, columnar striated epithelium, lamina propria, and microvilli making the function of the stomach specialized. ese findings are in agreement with those of [27,28]. In this study, it was observed that the stomach muscularis possesses a circular layer    that is thicker than the longitudinal layer, which is consistent with the findings of [22,26,29]. e small intestine, which is the anterior section of the intestine, showed numerously mucus glands; the mucosa revealed longitudinal folds of finger-like projections (villi), with a prominent columnar epithelium; and the macro-and microvilli possess cells that line the entire length of the intestine. e provision for excess mucus gland aids lubrication and easy passage of food materials, protecting the mucosal epithelium from mechanical or chemical injuries arising from interactions with digestive tract contents or enzymes, and the presence of absorptive cells helps in the absorption of valuable nutritive substances. e villi help to absorb nutrients from the food eaten and then shuttle the nutrients into the bloodstream of every species [30][31][32]. e posterior region which is the large intestine shows gastric pits and glands, the mucosal folds are numerous, and the presence of large villi is prominent. e columnar epithelium is distinct. e large intestine has a thick and dense circular muscle, the submucosa has very reduced gastric glands and gastric pits, the columnar epithelium and lamina propria are short (Figure 8(e)). e cloaca is packed with dense circular muscle and a reduced submucosa (Figure 8(f )). e cloaca is a region for reserving waste before it is passed off the gut. e cloaca has strong muscle for appropriate contraction to propel defecation.
e Heterotis species are available all year round in large numbers. In this study, it was noticed that size was related to sex (Table 1). Using the mean and standard deviation, it was seen that female Heterotis species were larger than the males with a significant difference. A t-test was used to compare sex to size (length and weight). e result of the t-test of length and weight of females was 0.6, while for males 0.1 was obtained. Females are possibly larger than males because of the tendency to acquire food in preparation for reproductive capacity, hence the need for extra capacity and the ability of the anatomy and body structure to accommodate reproductive organs. Furthermore, females could grow larger because they possibly eat more for extra energy during spawning, hatching, and care of young ones. e sexual abundance (1 : 1.5) in favor of the females in the Lower Niger River may be influenced by variable factors including food availability, short lifespan, rapid growth, and conducive environment. Variety of fish species in addition to other macro-and microinvertebrates is available all year round in the river, thus enabling rapid growth and high recruitment. e active survivors prey on the rich variety of food available in the river; they grow very fast and become recruited into the fishery. e length-weight relationship (LWR) of Heterotis niloticus obtained in the study for the whole population (both males and females) revealed a correlation coefficient (r) of 0.83 as shown in Table 2. e value obtained for b was 0.53 for the whole population, 0.41 for males, and 0.26 for females. is result shows that H. niloticus exhibits a negative allometric growth (b < 3.0) meaning that the length increases much more with body weight. is finding is in line with those of [33,34,35]. e length-girth relationship of H. niloticus showed a strong and positive correlation coefficient (r) of 0.77. e mean condition factor K was 1.51 (t-test, P > 0.05), which indicates that the Heterotis are in good condition in the river. Many factors could determine the productivity of a river; availability of food, proper management policies, pollution, turbidity, and indiscriminate dredging could affect the condition and production of the river. Moreover, heavy bodied fishes such as the Heterotis species are noted to have higher condition factors than those with flattened bodies [36]. It is also reported that H. niloticus can thrive well in polluted and turbid waters, hence its ability to maintain a condition factor >1 [37,38]. Out of the 125 specimens obtained, 36 were males while 53 were females with a ratio of 1 : 1.5, and the remaining 36 were unsexed. All of these specimens were examined for variety of food items; 69 had empty stomachs while 56 had food items in their stomachs. e percentage composition of food items obtained from H. niloticus from November 2019 to October 2020 is shown in Table 3.
e dominant food items found were copepods (33.48%) and shrimps (16.74%) by %FO. Other food items of low importance were mollusc, plant parts, sand, and detritus: 14.16%, 12.02%, 3.43, and 14.59%, respectively (Table 3). e food item with the most relevance was copepod with a percentage of 34.45%. e food items obtained in this study from the stomachs of H. niloticus show a dominance of arthropods and crustaceans in addition to detritus materials, suggesting frequent   Table 3. It is evident that H. niloticus was strictly a planktonic microphage. Larger fish had some whole fish or sometimes fish flesh in their stomach. e presence of plant remains and sand could be due to the probability that while grazing on other benthic organisms it scraped, quickly pulled out, or nipped off plants along with their substrate. Hence, the variation of food items, which include sand, detritus, fish, copepods, shrimps, and mollusc, makes it possible to conclude that H. niloticus is an omnivore. From the shape of the mouth and the arrangement of the gills, it could be concluded that filter-feeding habit is made possible by the possession and arrangement of fine gill rakers and gill filaments as seen in Figures 4 and 5.
Although the species had earlier been described as more of a plankton feeder [11,39], this study has shown that H. niloticus is an omnivore with arthropods and crustaceans being the significant food items in its diet with the capability of filtering planktons and other food substances in water. Similar findings proving that H. niloticus is an omnivore were reported by [34,[39][40][41]. e nature of the gut, possession of a gizzard (food grinder), and long gut length (extended absorption) of the intestine are important features that show omnivorous feeding. e gizzard serves as an organ of digestion. Fluid and digested matter were preset in the gizzard with no grains or seeds as in the case of birds. e gut length recorded in this study (mean of 77.32 cm) and the relative gut length (RGL) of 1.08 suggest a long gut transit time for the food of this fish. According to Biswas [16], fish can be classified as herbivorous (RLG >1), carnivorous (RLG <1), or omnivorous (RLG �, >, or <1). With regard to the RGL, the result obtained in this study shows that H. niloticus is a herbivore, but the food items discovered do not show that. Following the theory of Ward-Campbell and Beamish [42] which is supported by [43][44][45][46], it is generally accepted that RGL< 1 indicates carnivorous diet, and RGL < 3 > 1 indicates omnivorous, whereas values of RGL ˃3 indicate diet based on plant material or detritus. is theory is an added proof that H. niloticus is an omnivore. In this study, the prey-predator relationship of H. niloticus revealed that fish weight was more related to prey size as shown in Figures 9 and 10.
A linear and positive relationship between the predator body weight and prey body weight of H. niloticus was observed. Larger prey was obtained as the fish size (weight) increased. A perfect relationship between the weight of H. niloticus and its prey was obtained. is shows that as the fish gets bigger, larger sizes of meals were preferred to smaller ones; hence, size of fish is associated with feeding capacity. Similar findings relating feeding capacity to size were reported by [47][48][49][50][51][52].
Heterotis niloticus possesses a single ovary located on the right side. Color of ovary is amber or ovary pink at the quiescent/immature stage, deep pink in mature stages, and yellow to orange in mature and running stages. e ovary is elongated and larger at the anterior part and thinner at the posterior end towards the cloaca, laterally compressed, and has 51-82 ± 68 lamellar folds. H. niloticus showed variation in gonadosomatic index (GSI) which was greatly induced by season and size of fish. Monthly averages of GSI were recorded in the rainy season with its peak in the months of May to September and then a decrease till November. No gravid females were found in November till early February, which confirms that the spawning period begins as the flood plains rise in the rainy season. e African bonytongue has relatively large eggs (2.3 mm-3.00 mm) and moderate fecundity (2100-32000 eggs) (Table 4). e size of H. niloticus in the Lower Niger River at first maturity was not calculated in this study because the size of sample collected is small (<500). e number and size of eggs were dependent on the size of fish. e weight in relation to its length was a determinant for ovarian weight and oocyte count available to a species. Mature fishes represented gonad stages of 2-5. Mature fishes were recorded from fish with sizes of 368 g-2000 g. e smallest mature fish observed has a TL of 31 cm, SL of 21 cm, and W of 368 g, with the length of the gonad being 5.3 cm and weight being 5 g. Fecundity was obtained only for 19 fecund female specimens.
Six stages of gonad development were found in H. niloticus during the sample period in both males and female fishes: the immature, quiescent, maturing, mature, running, and spent stages (Table 5). e decline in rainfall which correlated to reduction and absence of gravid females suggests the end of spawning periods. e changes in GSI revealed that H. niloticus has specific periods for maturation of ovaries, spawning, and breeding times. H. niloticus is thus observed to be  moderately fecund ((2100-32000 eggs) when compared to fish species like the African mudfish (Protopterus annectens) and the grunt (Pomadasys jubelini) [2,53]. ese results are in conformity with the findings of [19]. is study reveals an overlap in the ranges of average sizes in relation to maturity stages. is could be because H. niloticus does not depend on length alone but also weight of the fish and other contributing factors such as availability of food, water quality, climate changes, and neural responses [54][55][56][57][58].

Conclusion
Heterotis niloticus is an omnivorous macrophage detritor, consuming a wide variety of bottom-dwelling food items. e anatomy of the mouth and gut along with its histology is modified to accommodate the feeding habits. H. niloticus has a single ovary which rests on the right side of the fish; it is reproductively active as the flood plains rise and peaks at the peak of the rains, being a moderately fecund fish. e high fecundity is complemented with peaks of GSI observed during the months of September, October, and November, which reveals spawning periods. Hence, the aforementioned attributes of the African bonytongue make it a suitable candidate for culture.

Abbreviations
H. niloticus: Heterotis niloticus GIT: Gastrointestinal tract N: Sample size Df: Degree of freedom.    (19) Data Availability e data used to support the findings of this study are included in the article.

Ethical Approval
Not applicable.

Consent
Not applicable.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this study.

Authors' Contributions
Dr. M. O. A. is the principal researcher and was responsible for sample collection, analyses, and manuscript writing. Mr. F. E. A. participated in handling field and laboratory procedures. Dr. H. O. E. contributed to histological preparations and analyses of processes. All authors read and approved the manuscript.