Effect of age on the structure and activity of the pharynx of the free-living nematode Metarhabditis andrassyana (Rhabditidae)

The study of the pharynx in the nematode Metarhabditis andrassyana (Tahseen et al. in Int J Nematol 14:163–168, 2004) revealed that the activity of pharynx declined with age and was also reduced in the absence of E.coli. A comparison between presence and absence of bacteria in the same age group revealed a reduction of pharyngeal pumping in both 2-day-old individuals and 8-day-old individuals. However, when nematodes were placed together in groups of varying numbers, pharyngeal pulsations declined as the numbers of specimens increased from 5 to 15 in the presence or absence of bacteria as a food source with age. In old worms, abnormalities were observed in the structure of the pharynx. Various structural changes occurred on the surface of the pharynx as well as in the lumen, tissues and the grinder. The deteriorative changes in the basal bulb reveal an involvement of almost all regions of the bulb—the outer surface, muscles and the grinder.


Introduction
The pharynx in nematodes connects the stoma or feeding apparatus with the intestine. The morphology of the pharynx varies depending on the feeding habits of the nematodes. The overall structure may be divided into two or more parts and is essentially muscular so that the food taken in can be forced into the intestine. In most nematodes it works as a pump to force the food into the intestine against internal turgor pressure (Harris and Crofton 1957;Bennet-Clark 1976;Avery and Shtonda 2003). Pharyngeal pumping is accomplished by sequential contraction of muscle fibers that creates a wave of dilation which propagates along the lumen from the anterior to the posterior region (Fürst von Lieven 2003). In most rhabditid nematodes the pharynx shows two basic activities: pumping and isthmus peristalsis (Fig. 1). The pumping cycle that involves the corpus, anterior isthmus and terminal bulb begins by contraction of muscles that open the lumen in these regions. During isthmus peristalsis contraction of muscles pulls the lumen open in the posterior isthmus and transports the food into the terminal bulb (Doncaster 1962;Seymour et al., 1983;Avery and Horvitz 1989;Chiang et al. 2006). During the pumping phase the terminal bulb is isolated from the corpus and anterior isthmus because the posterior isthmus is closed (Avery and Shtonda 2003). Peristalsis in the posterior isthmus is delayed and occurs after approximately every four pumps (Avery and Horvitz 1987). Pharyngeal activity like the other body functions, declines as the nematodes age. This reduction in pharyngeal activity may be caused by several factors. In Caenorhabditis elegans, aging results in structural deterioration or sarcopenia of the pharynx muscles and is considered the primary cause of reduced pumping cycles (Johnson 1987;Garigan et al. 2002;Herndon et al. 2002;Glenn et al. 2005). It was also confirmed that aging produced no abnormal changes in the neurons of the pharynx (Herndon et al. 2002), suggesting that there was no nervous involvement in reduced pharyngeal activity. Laser ablation of identified neurons innervating the pharynx also revealed that, even in the absence of all pharyngeal neurons, pumping continues and only one pharyngeal neuron, M4, is essential for feeding and hence for life (Avery and Horvitz 1987;Avery and Horvitz 1989;Avery 1993). The pharynx of M. andrassyana is the typical tripartite type, viz., it is possible to see the procorpus, a narrow isthmus (anterior isthmus is the upper part of isthmus connected with corpus while posterior isthmus, connected with the basal bulb), and a basal 1 3 bulb with grinder. The aim of the study is to understand the mechanism of the two feeding motions and the effect of age on the structure and activity of the pharynx in M. andrassyana.

Sampling
Farmyard manure samples collected near the fort (27.8°N, 78°E) in Aligarh were processed by the sieving and decantation and the Baermann's funnel technique (Flegg 1967).

Culturing the nematode
Metarhabditis andrassyana was cultured in the laboratory on Nematode Growth Medium (NGM). First start the seed culture; M. andrassyana culture was initiated by transferring nematodes collected from manure into plain 1% water agar supplemented with milk powder. A single gravid female from the seed culture was transferred into a 0.1% streptomycin sulfate solution, and an incision was made in the posterior region. The cut caused the release of eggs into the streptomycin sulfate solution. The eggs were left for five minutes and then repeatedly washed in sterile water. A monoxenic culture was initiated by transferring the sterilized eggs into 5-cm Petri dishes containing NGM which had been inoculated with E.coli (OP50) and left overnight at room temperature to allow bacteria to multiply.

Isolation of different age groups of males and females
Similar age groups of males and females of M. andrassyana were obtained by transferring developing J4 stage males and females on to separate NGM plates. The nematodes were harvested at the required age.

Feeding
Five young nematodes were placed on a 15 × 10 × 2 mm strip of NGM agar with Escherichia coli bacteria on a glass slide. A coverslip was gently applied. The nematodes were allowed to acclimatize for 5 min after which pharyngeal pumping was observed and photographed on an Olympus BX 50 DIC microscope.

Pharyngeal activity
Pharyngeal activity which typically has two phasespumping and posterior isthmus peristalsis-was studied in both individual specimens and multiple specimens in groups of varying numbers.
(i) Single specimens Single specimens of two age groups were studied, 2-day-old and 8-day-old nematodes. Further, each set of nematodes was observed in the presence and absence of the bacteria (E.coli). A 15 x 10 x 2 mm strip of NGM agar with or without bacteria was placed on a glass slide, and a single individual was placed onto it. A coverslip was gently applied. The nematode was allowed to acclimatize for 5 min after which pulsation and isthmus peristalsis were observed and recorded on a Zeiss Discovery V20 stereo microscope. There were twenty replicates using equal number of females and males for each set, i.e., with bacteria and without bacteria. The observation time was two minutes. The same protocol was followed with the 8-day-old individuals. (ii) Multiple specimens When multiple specimens in groups of varying numbers were used, only pharyngeal pulsation was observed. Groups of 5, 10 and 15 specimens were studied. In each group, 2-day-old and 8-day-old specimens were observed separately. For each of the groups of nematodes (two age groups with three size groups each, respectively), pharyngeal pumping activity was recorded both in the presence and absence of bacteria. All these specimens were studied on NGM agar plates, and pumping was recorded for 2 minutes on a Zeiss Discovery V20 stereo microscope. There were ten replicates, five of males and five of females, for each set.

Pharynx structural changes
In total, 10-20 nematodes of 2-day and 8-day age groups were mounted individually on 2% agarose pads and paralyzed with alcohol (Wu et al. 2019). The pharynx was studied and photographed using an Olympus BX 50 DIC microscope with a mounted ProgRes C3 camera.

Statistical analysis
All the data were statistically analyzed by one-way ANOVA using GraphPad Prism 7 to reveal significant difference between mean values of different age groups. All values are presented as mean ± deviation (SD). The probability levels of 0.05 were considered statistically significant.

Feeding
The bacterial food source is ingested via the stoma in M. andrassyana. The lumen of the pharyngeal tube communicates with that of the stoma, even when the rest of the pharyngeal lumen is closed. The anterior end of the pharynx contains the corpus, which brings about ingestion during muscle contraction. The posterior end of the pharynx contains the terminal bulb, which crushes the bacteria in the grinder so they can be digested in the intestine. Pharyngeal muscle contraction opens the lumen and relaxation closes it. During pumping, it is necessary for different parts of the pharynx to contract to effectively transport food to the intestine. A pharyngeal pump begins with the nearly simultaneous contraction of the corpus and the terminal bulb resulting in the drawing of food particles into the pharyngeal lumen. This is followed by a contraction of the anterior isthmus. The anterior part of the corpus relaxes first, then the posterior part of corpus which is adjacent to the isthmus. The bacteria ingested are then trapped in the anterior isthmus. Posteriorly propagating contractions of the posterior isthmus, known as peristalsis, transport bacteria from the anterior isthmus to the terminal bulb. Peristalsis in the posterior isthmus occurs every 4-5 pumping cycles (Fig. 2). In the basal bulb, at the beginning of grinding process, the open passage between terminal bulb and intestine allows fluid flow from the gut toward the grinder. The contraction of the lumen of terminal bulb pushes particles out of the haustrulum and into the intestine.

Pharyngeal activity
(i) Single specimens Pharyngeal activity declined with age and was also reduced in the absence of E.coli. Pumping activity declined significantly (p < 0.05) by 68% and isthmus peristalsis by 73% in 8-day-old nematodes as compared to 2-day-old specimens, in the presence of bacteria. In the absence of the food source the decline was even greater with pumping being reduced by 73% and peristalsis by 78%. A comparison between presence and absence of bacteria in the same age group revealed a reduction of 27% for pumping as well as peristalsis in 2-day-old individuals, while in 8-day-old individuals the reduction of 39% for pumping as well as peristalsis between presence and absence of bacteria (Fig. 3). All declines were significant (p < 0.05) F (3, 72) = 4621 p < 0.0001. The average number of pulsations per peristalsis remained relatively constant between 4 and 5 pulsations/peristalsis. The average number for 2-day-old individuals was 4.1 with and without bacteria and 5.0 with bacteria and 4.7 without bacteria in 8-day-old individuals.
(ii) Multiple specimens When nematodes were placed together in groups of varying numbers, pharyngeal pulsations declined as the numbers of specimens increased from 5 to 15 in the presence or absence of bacteria as a food source in two-day-old nematodes (Fig. 4). In eight-day-old nematodes, pumping activity was greatly reduced as compared to 2-day-old nematodes and it declined further with increase in numbers both with and without a food source. In 2-day-old nematodes in the presence of E. coli the decline in pumping rate of the 15 nematodes group was with 17% significantly lower than the rate of the 5 nematodes group. In the absence of bacteria, the difference between the said two groups was even greater (29%). In 8-day-old nematodes the reduction in pulsations was significant (p < 0.05) F (6, 108) = 44.98 p < 0.0001 at 26% in the presence of bacteria and only 9% in absence of bacteria between 5 nematode and 15 nematode groups (Fig. 4). In all 2-day-old nematode groups pharyngeal pumping was significantly higher (p < 0.05) in the presence of bacteria than in its absence. The reduction varied from 27% in the 5 nematode group to 38% in the 15 nematodes group. In the 8-day group significant reduction of 22% and 26% occurred between the 5 nematode and 10 nematode group and was only 4% in the 15 nematode group (Fig. 5A). In the presence of bacteria when groups of similar numbers were compared between young and old individuals, then there was a drop in pulsation rate from 71% in the 10 nematode group to 75% in the 15 nematode group. Without food the pumping rate was reduced to 61% in the 15 nematode group, 69% in the 10 nematode group and 70% in 5 nematode group (Fig. 5B).

Pharynx structural changes
With age several structural changes occurred on the surface of the pharynx as well as in the lumen, tissues and the grinder. Shrinkage and distortion appeared on the outer lining on some parts of the procorpus (Fig. 6C) and the basal region of the basal bulb (Fig. 6D). The lining becomes thinner and depressed on the procorpus and appears to shrink inward in the basal bulb distorting the smooth and uniform outline. The distorted outline of the basal bulb is also visible in Fig. 6E, F. The internal changes were generally more pronounced. In some specimens the metastegostomal region became swollen with a large vacuolated appearance (Fig. 6A). Vacuolated areas also appeared in the corpus (Fig. 6C) and basal bulb (Fig. 6E). Vacuolated areas showed a hyaline structure and appeared to be filled with fluid. There was no sign of muscular tissues in the vacuoles, suggesting that it may have been reabsorbed. In addition, the procorpus lumen often became distorted and irregular (Fig. 6B). The basal bulb occasionally became elongate with a disorganized grinder (Fig. 6G). In many nematodes the haustrulum muscles near the base of the bulb were difficult to visualize indicating degenerative changes (Fig. 6F).

Discussion
In most rhabditid nematodes, a pharyngeal contraction opens the lumen and draws food in while relaxation closes the lumen and traps and transports the food down toward the  (Avery and Shtonda 2003). A slight delay in the relaxation of the anterior isthmus makes it possible to receive and accumulate the bacteria that have passed down from the anterior corpus. Like in C. elegans (Seymour et al. 1983;Avery and Shtonda 2003) bacteria tend to accumulate at the base of the stoma before entering the pharyngeal lumen at the end of the contraction cycle. Regurgitation of intestinal contents into the basal bulb was not observed as in several rhabditid nematodes, viz., Poikilolaimus oxycercus, Caenorhabditis elegans (Fürst von Lieven 2003), but the possibility of this happening may not be ruled out.
Metarhabditis andrassyana showed the general tendency of decline in the pharyngeal pulsations with age when studied individually. Similar observations have also been made in C. elegans (Bolanowski et al. 1981;Kenyon et al. 1993;Huang et al. 2004). Besides age, availability of food also appeared to influence the pharyngeal pumping as within the same age group pulsations declined significantly in the absence of bacteria. Croll and Smith (1978) and Horvitz et al. (1982) suggested that bacteria stimulated pharyngeal pulsation, a phenomenon that may also be operating in M. andrassyana. When nematodes were studied in groups, it becomes clear that numbers also influence the rate of pulsations in the presence of food or without food. In 2-day-old nematodes the decline was significant between the three different numbers of specimen groups tested. Although pulsations were occurring significantly rarer in the absence of food than when food was provided, the differences between these groups also varied significantly. In the 8-day-old nematodes pulsations differed significantly only between two groups. The rate of decline in the absence of food came down to just 4% in the 15 nematode group, while it was maintained above 20% in the other two groups. This near similarity in the rate of pulsations in the absence of food in the 15 nematode group may represent a basal rate, which is just sufficient for survival. In young 2-day-old nematodes, the significant decline in pharyngeal pumping in the presence of bacteria perhaps indicates that secretions of the nematodes together with excrements and other metabolic waste products may also be playing a role in the pulsation rate via a chemo-sensory pathway. If contact with one another is also considered as a probable cause, it may represent a thigmotactic response affecting the pharyngeal pumping.
The gradual degenerative changes in the structure of the pharynx are correlated with pharyngeal pulsation and synchronous with its decline in aging individuals. The swelling and vacuolation of the corpus adjacent the metastegostom not only deforms the stegostom but in all probability also affects the process of ingestion and may be one cause of bacteria clogging the stoma. The distortion of the luminal lining, a slight shrinkage/narrowing of the corpus wall and the vacuolation in the basal region of the corpus could affect the transport of food down the pharynx. All these changes in the anterior pharynx may not affect pulsations per se but could certainly influence ingestion via metastegostomal disorganization and transport because of luminal distortion and vacuolation of corpus tissue and could result in accumulation of bacteria in the pharynx. Chow et al. (2006) suggested that bacterial plugging was a consequence of reduced pumping and not the cause as suggested by Garigan et al. (2002).
Our observation of the deteriorative changes in the basal bulb reveals an involvement of almost all region of the bulb-the outer surface, muscles and the grinder. The most spectacular changes are in the muscles in all parts of the bulb that may severely impair the pumping mechanism. Further the distortion of the grinder, brought about by muscle degeneration or mechanical damage caused by continuous pumping, may also be a cause of bacterial plugging of the basal bulb.
Acknowledgements The financial assistance provided by the University Grants Commission (UGC) to the first author (HF) is gratefully acknowledged.
Author contributions HF conducted the experimental work and wrote the manuscript, while IA helped to develop the study design and with manuscript editing.  Fig. 6 Pharynx of aged nematodes; A vacuole at the base of stoma in older worms; B irregular lumen of pharynx in aging; C degeneration in corpus (filled right arrow) and vacuoles present (unfilled right arrow); D irregular basal bulb wall (filled right arrow); E large vacuole in basal bulb (unfilled right arrow) and muscles degeneration (filled right arrow); F degenerated muscle at the region of basal bulb (filled right arrow); G structure of grinder changed and became asymmetrical (filled right arrow). Scale bar: A-G = 10 μm