Circadian variation in the metabolic activities of the American cockroach, Periplaneta americana (Dictyoptera: Blaatidae) found in Abeokuta, Nigeria

Concilia Ifesinachi IYEH, Kehinde Olutoyin ADEMOLU,Adebola Adedoyin OSIPITAN, Adewumi Babatunde IDOWU,Abiodun Oladipupo JODA

(1.Department of Animal and Environmental Biology, Federal University, 371104 Oye-Ekiti, Nigeria;2.Department of Pure and Applied Zoology, Federal University of Agriculture, 111101 Abeokuta, Nigeria;3.Deaprtment of Crop Production, Olabisi Onabanjo University, 120107 Ayetoro Campus, Nigeria)

Abstract：【Aim】 Periplaneta americana commonly known as the American cockroach carries out a wide range of activities with the help of locomotor abilities using its femoral and thoracic muscles, reflecting ease of movement in various habitats and that it is able to utilize food substances by the help of metabolic activities for effective daily activities. Little is known about variations in the daily pattern of metabolic activities in P. americana. This study was conducted to investigate the circadian variation in metabolite contents and enzyme activities in the thoracic and femoral muscles of adult P. americana. 【Methods】 The study was carried out by collecting the femoral and thoracic muscles of the 8-day-old aduts of P. americana at 6:00, 12:00, 18:00 and 24:00 local time [6:00, 12:00, 18:00 and 24:00 Greenwich mean time (GMT)]. The activities of four enzymes (amylase, lipase, proteinase and α-glucosidase) which are responsible for metabolizing or converting the food substances to utilizable substances during transport to the muscles and hydrolysis of the food substances necessary for fueling locomotion in insects, and the contents of the organic (glucose, protein and lipids) and inorganic [sodium (Na+), potasium (K+), calcium (Ca2+), chlorine (Cl-), iron (Fe2+), magnesium (Mg2+) and phosphate ion substances in these tissues were determined using standard biochemical analytical methods.【Results】 The activities of the four enzymes amylase, lipase, proteinase and α-glucosidase were detected in both femoral and thoracic muscles of P. americana adults throughout the 24 h but at varying activity levels at different time. The activities of amylase, lipase, proteinase and α-glucosidase tested in the femoral muscle followed this pattern: 12:00>6:00>24:00>18:00, and were higher in females than in males. In the thoracic muscle, the activities of amylase, lipase and proteinase at 6:00 and 12:00 were significantly higher than those at 18:00 and 24:00, and females had higher enzyme activities than males. In the thoracic muscles of adult males, the glucose content was significantly higher at 18:00 than at the other time points, and the protein contents at 18:00 and 24:00 were significantly higher than those at 6:00 and 12:00, and the lipid content was significantly higher at 24:00 than at the other time points, while in the thoracic muscles of adult females, the lipid content was significantly higher at 12:00 than at the other time points, the protein contents at 12:00 and 24:00 were significantly higher than those at 6:00 and 18:00, while the glucose content was significantly higher at 24:00 than at the other time points. In the femoral muscle, the lipid content was significantly higher at 18:00 than at the other time points. In the femoral muscle of adult males, the glucose content was significantly higher at 24:00 than at the other time points, and the protein content was significantly higher at 6:00 than at the other time points, while in the femoral muscle of adult females, the contents of glucose and protein were significantly higher at 24:00 than at the other time points. In the thoracic muscle of adult males, the Na+ content was significantly higher at 18:00 than at the other time points, the contents of K+ and Ca2+ in males at 6:00 and 12:00 were significantly higher than those at 18:00 and 24:00, the Cl- content was significantly higher at 24:00 than at the other time points, the contents at 6:00, 18:00 and 24:00 showed no significant difference, but were significantly higher than that at 12:00, while in the thoracic muscle of adult females, the Na+ content was significantly higher at 6:00 than at the other time points, while the contents of K+, Ca2+ and Cl- were significantly higher at 12:00 than at the other time points, and the contents at various time points were not significantly different. In the femoral muscle of the adult males, the contents of Na+ and Ca2+ were significantly higher at 12:00 than at the other time points, the K+ content was significantly higher at 6:00 than at 18:00 and 24:00, while the contents of Cl- and were significantly higher at 6:00 than at the other time points, while in the femoral muscle of adult females, the contents of Na+ and Cl- were significantly higher at 6:00 than at the other time points, the contents of K+ and Ca2+ at 6:00, 12:00 and 18:00 exhibited no significant difference, while were significantly higher than that at 24:00, and the content exhibited no significant difference among various time points. 【Conclusion】 The results of this study suggest that P. americana adults possess the ability to metabolize their organic and inorganic substrates with the help of the enzymes amylase, lipase, proteinase and α-glucosidase for movement, during the peak time of activities.

Key words：Periplaneta americana; enzymes; femoral muscle; thoracic muscle; metabolic activity; organic substance; inorganic substance

1 INTRODUCTION

American cockroach also known asPeriplanetaamericana, is an invertebrate animal which belongs to the Insecta class, Dictyoptera order and Blattidae family. It is known as one of the largest, strongest, oldest, and most successful breeding insect groups (Shi, 2012). Cockroaches exhibit a range of locomotor abilities, reflecting ease of movement in various habitats. American cockroaches are omnivorous in nature and feed on various kinds of foods (Bell and Adiyodi, 1981). They possess digestive system which produces various kinds of enzymes that break food substances for maintenance, survival and reproduction. These food substances are broken into absorbable form required for metabolic activities in the body of insects (Oluwakemietal., 2014). The occurrence of a multitude of digestive enzymes in the gut of cockroaches is consistent with their omnivory and feeding adaptability (Oluwakemietal., 2014).

Thoracic and femoral muscles play a major role in the dispersive or locomotor abilities of insects, the survival of insects depends on their abilities to run and fly which are facilitated by these two tissues. Locomotor activities in insects depend on many factors, including diet, which have been reported to have a significant effect on the flight activities of insects as they are influenced by the enzyme activities in both femoral and thoracic muscles (Ademoluetal., 2013). Many substrates are metabolized for dispersal activities by insects suggesting the presence of their enzymes (Chapman, 1990). Bertram (1955) had earlier reported that food substances were converted to utilizable substances prior to or during transport to the muscles. Yacoeetal. (1982) has also reported that diets affect the oxidation and metabolism of the substrate in the muscles. Insects consume certain foods which are metabolized into fuel for the active tissues concerned with locomotion and also these foods have been reported to play a significant role in the locomotor activities of insects by providing energy needed for the flight tissues (Ademoluetal., 2013). The enzyme activities in the femoral muscles have been reported to increase asZonocerusvariegatusdeveloped from the 1st nymphal instar until the adult stage (Ademoluetal., 2009). The activities of enzymes and the contents of metabolites that influence the diets to supply energy for locomotor activities of these muscles of cockroaches have been scarcely reported. Circadian rhythms of physiological functions and behavioural patterns are normally displayed in living organisms to adapt and synchronize the organisms with periodic changes in the environment (Ishidaetal., 1999). The activities of invertebrates are controlled by exogenous factors (Grimm and Schaumberger, 2002) as well as internal circadian and circannual rhythms. Reports have shown that the peak level of locomotor activity of some invertebrates occurred at night during 23:00-24:00, while the lowest level of locomotor activity occurred during 10:00-17:00. The habit of going out at night is an adaptive mechanism in invertebrate animals (Ademoluetal., 2011). Adult forms of hemimetabolous insects, particularly cockroaches, have been utilized extensively as model systems for the investigation of the physiological basis of circadian rhythmicity (Page, 1990). Less attention has been paid to the development of the circadian system in this group of organisms. Circadian clocks are known to control rhythms in physiology and behaviour entrained to 24 h light-dark cycles, locomotor rhythms are the most prominent overt rhythm controlled by the clock (Tomioka and Matsumoto, 2019) and circadian system controls locomotor activity inP.americanavia Melatonin receptor 2 (MT2) (Kamruzzamanetal., 2021). Previous reports have shown that the circadian clock plays an important role in various physiological processes, and regulates daily activity rhythms, the timing of mating behaviour, time-of activitie day-related memory, sun-compass-dependent orientation or navigation, and photoperiodic time measurement for seasonal adaptation (Saundersetal., 2002). Circadian variations in metabolic activites and the pattern of daily enzyme activities in the femoral and thoracic muscles (the tissues responsible for locomotion) ofP.americanahave not received any attention in the literatures. Thus, the aim of this study is to investigate the daily variation in the metabolic activities of the femoral and thoracic muscles ofP.americana.

2 MATERIALS AND METHODS

2.1 Test insects

This experiment was carried out at the laboratory of the Department of Pure and Applied Zoology, College of Biosciences, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria. Nymphs of undetermined instar of the American cockroach (P.americana) were obtained from students’ cupboards in Federal University of Agriculture Abeokuta, Ogun State, student hostels and households around Funaab. Locally made traps were placed in wardrobes, kitchen cupboards, bins in the study area. The trapped nymphs were kept in plastic vials, which were smeared with vaseline; to help slow down cockroaches from running out of the vials, since they possess cursorial legs. The traps were also covered with a muslin cloth and a rubber band attached to hold it firm. As soon as they moulted into adults, they were separated into males and females according to their ages and about 8-day-old adults were taken to the laboratory, where chemical analysis was carried out. The daytime was divided into four time points: 6:00, 12:00, 18:00 and 24:00 local time [6:00, 12:00, 18:00 and 24:00 Greenwich mean time (GMT)]. Thirty individuals ofP.americanaat per time point were taken out of the plastic vials and their muscles were carefully dissected out. All experiments were replicated three times during this study.

2.2 Femoral muscle collection

A total number of 30 adults (15 males and 15 females) were randomly picked, their hind femurs were dissected, and all the muscles were removed with forceps into a test tube and homogenized in 0.05 mol/L KCl. The homogenates were centrifuged at 500 r/min at 5 ℃ for 30 min. The supernatants were collected in a 30 mL centrifuge bottle and stored in a deep freezer until further analysis (Ademoluetal., 2013).

2.3 Thoracic muscle collection

A total of 15 male and 15 female adults were randomly picked, their thoracic muscles were dissected and removed as described in Modder (1984). The thoracic muscles were separated from the fat body and rinsed with water and later homogenized in distilled water. The homogenates were centrifuged at 500 r/min at 5 ℃ for 15 min. The supernatant was collected in a bottle and stored in a freezer at 30 ℃ for further processing (Ademoluetal., 2013).

2.4 Analysis of enzyme activities in P.americana adults

2.4.1Analysis of amylase activity: Two millilitres of the enzyme extracted in a 1 mol/L potassium hydrogen phosphate buffer (pH 6.9) were mixed with 1 mL of 1% starch solution and incubated for 1 h. The reaction was stopped by adding 8 mL of DNSA reagent. The mixture was boiled in a water bath for 5 min, cooled in a cold water bath and then diluted with 18 mL distilled water. The absorbance of the resulted solution was read at 550 nm on an SP 6250 Spectrophotometer (Ademolu and Idowu, 2011).

2.4.2Analysis of a-glucosidase activity: 0.5 mL of the enzyme extract was mixed with 1 mol/L of para-hydroxy benzoic acid, 5 mL of 0.1 mol/L sodium phosphate buffer at pH 5.5 was added, followed by 1 mL of distilled water. DNSA reagent (1 mL) was added to the mixture, which was heated at 100 ℃ for 5 min, then rapidly cooled to room temperature followed by the addition of 10 mL of distilled water. A standard solution of glucosidase enzyme was prepared and treated similarly. The absorbance of sample extract and glucosidase standard solution was read on a Cecil 600 Spectrophotometer at the wavelength of 530 nm (Ademolu and Idowu, 2011).

2.4.3Analysis of lipase activity: The extraction buffer was 0.2 mol/L sodium acetate-acetic acid mixture at pH 5. A 7.5 mL sample of the enzyme extract was added to the reaction mixture containing 0.4 g of sodium taurocholate, 1 mL of 0.1 mol/L CaCl2and 6 mL of the sodium acetate buffer. The mixture was incubated at 35 ℃ for 1 h, then the reaction was stopped by adding 40 mL of absolute alcohol. A standard lipase solution was also prepared using distilled water. The absorbance of the sample and standard was read at a wavelength of 415 nm on a Cecil 600 Spectrophotometer (Ademolu and Idowu, 2011).

2.4.4Analysis of proteinase activity: The extraction buffer was 0.1 mol/L sodium hydrogen phosphate (pH 6.9); 5 mL of the enzyme extract was added to 10 mL of 2% casein solution and incubated at 35 ℃ for 30 min. The reaction was terminated by adding 10 mL of 10% trichloroacetic acid solution and then filtered through Whatman No.1 filter paper. Standard proteinase solution was prepared and treated as above. The absorbance of the sample and standard proteinase filtrates was read at a wavelength of 275 nm. Proteinase activity was measured as the amount of proteinase capable of catalyzing absorbance change of 0.01 in 60 s at a wavelength of 275 mm (Ademolu and Idowu, 2011).

2.5 Determination of the contents of organic substances

The protein contents in femoral and thoracic muscles were determined by the method of Henryetal.(1974), while the Baunmniger (1974) method was adopted for glucose determination. Lipid assay was done by the method of AOAC (1990).

2.6 Determination of the contents of inorganic substances

2.7 Statistical analysis

Data were analyzed using the Statistical Package for Social Sciences (SPSS) version 20.0 (IBM Corp, 2011). Mean values were compared using analysis of variance (ANOVA). Results were presented as mean±standard deviation.Posthoctest was done using the Student-Newman-Keuls (SNK).P<0.05 was considered to be statistically significant.

3 RESULTS

3.1 The enzyme activities in femoral muscle

In the femoral muscle of the 8-day-old adults ofP.americana, the amylase activity at 12:00 was the highest, significantly higher than those at the other time points (P<0.05), followed by that at 6:00, and that at 18:00 was the least. It was observed that the amylase activity followed this pattern: 12:00>6:00>24:00>18:00. In the femoral muscle, the amylase activity in females was higher than that in males. The highest lipase activity was recorded at 12:00 and the lowest at 18:00. Females had higher lipase activity than males (P<0.05). In the femoral muscle, the proteinase activities at 6:00 and 12:00 were significantly higher than those at 18:00 and 24:00 (P<0.05), and females had higher proteinase activity than males. The α-glucosidase activity was significantly higher at 12:00 than at the other time points, and females had higher α-glucosidase activity than males at all the time points in the femoral muscle.

Table 1 Activities of four enzymes related to locomotion in the femoral muscle of the 8-day-old adultsof Periplaneta americana

3.2 The enzyme activities in thoracic muscle

In the thoracic muscle of the 8-day-old adults ofP.americana, the activities of lipase, amylase and proteinase at 6:00 and 12:00 were significantly higher than those at 18:00 and 24:00(P<0.05), and females had higher amylase and proteinase activities than males. In the thoracic muscles of adult males, the α-glucosidase activities at 6:00 and 12:00 were significantly higher than those at 18:00 and 24:00 (P<0.05), while in the thoracic muscles of adult females, the α-glucosidase activities at 6:00, 18:00 and 24:00 showed no significant difference, but were significantly lower than that at 12:00.

Table 2 Activities of four enzymes related to locomotion in the thoracic muscle of the 8-day-old adultsof Periplaneta americana

3.3 The contents of organic and inorganic substances in thoracic muscle

Table 3 showed the contents of organic substances in the thoracic muscle of the 8-day-old adults ofP.americana. In the thoracic muscles of adult males, the glucose content was significantly higher at 18:00 than at the other time points (P<0.05), and the protein contents at 18:00 and 24:00 were significantly higher than those at 6:00 and 12:00 (P<0.05), while the lipid content was significantly higher at 24:00 than at the other time points (P<0.05). In the thoracic muscles of adult females, the lipid content was significantly higher at 12:00 than at the other time points (P<0.05), the protein contents at 12:00 and 24:00 were significantly higher than those at 6:00 and 18:00 (P<0.05), while the glucose content was significantly higher at 24:00 than at the other time points (P<0.05).

Table 3 Contents of organic substances in the thoracic muscle of the 8-day-old adults of Periplaneta americana during 24 h

Table 4 Contents of inorganic substances in the thoracic muscle of the 8-day-old adults of Periplaneta americana during 24 h

3.4 The contents of organic and inorganic substances in femoral muscle

Circadian variations in the contents of the organic substances in the femoral muscle of the 8-day-old adults ofP.americanawere presented in Table 5. In the femoral muscle, the lipid content was significantly higher at 18:00 than at the other time points(P<0.05). In the femoral muscle of adult males, the glucose content was significantly higher at 24:00 than at the other time points(P<0.05), and the protein content was significantly higher at 6:00 than at the other time points (P<0.05). In the femoral muscle of adult females, the contents of glucose and protein were significantly higher at 24:00 than at the other time points(P<0.05).

Table 5 Contents of organic substances in the femoral muscle of the 8-day-old adults of Periplaneta americana during 24 h

Table 6 Contents of inorganic substances in the femoral muscle of the 8-day-old adults of Periplaneta americana during 24 h

4 DISCUSSION

Our study revealed that varying activities of amylase, α-glucosidase, lipase and proteinase were detected in the thoracic and femoral muscles of adult male and femaleP.americanaat different time points of the day during this study (Tables 1 and 2) and this agrees with the report by Ademoluetal. (2018). Modder (1981) detected and recorded similar enzyme activities in the thoracic muscles ofValangairregularis(Orthoptera: Acrididae). The presence of these enzymes in the locomotor tissues is an indication that hydrolysis of the organic substrates is necessary for fuelling locomotion in insects (Ademoluetal., 2018). The presence of these enzymes is an indication that these insects have the ability to metabolize their substrates as reported by Ademoluetal.(2009). Similarly, Chapman (1990) reported that many substrates were metabolized for movement by insects, suggesting the presence of their enzymes. Amylase has been reported to be essential in these two active tissues in order to hydrolyze stored glycogen to maltose and glucose which are assimilated in the body of insects (Ademoluetal., 2018).P.americanais a polyphagous insect consuming various food plants containing polysaccharides that require conversion to simpler carbohydrates before they can be utilized by the tissues (Toye, 1982).

The activities of amylase and lipase in the muscles of the 8-day-old adults ofP.americanawere significantly higher than those of other enzymes (Tables 1 and 2). The high amylase activity could possibly indicate high intake of carbohydrates which were readily available as earlier reported by Ademoluetal. (2009), carbohydrates when broken down produce energy for insects’ locomotor activities. Estela and Jose (2010) has also revealed that the first step required of fatty acid mobilization occurs in the action or ability of the fat body triglyceride lipases to catalyze the hydrolysis of the triglyceride molecules contained in the lipid droplets. Fatty acids are stored in the form of lipid droplets such as diglyceride, trehalose, or proline in the fat body, they are mobilized for the purpose of providing energy for flight muscles and also for the provision of lipids for ovaries and overall maintenance of the metabolic activities of other tissues, including the fat body.In the thoracic and femoral muscles of the 8-day-old adults ofP.americana, the activities of amylase, lipase and proteinase at 12:00 were significantly higher than those at the other time points (Tables 1 and 2), suggesting an increased locomotor activity at this time point and confirming that this time point is their peak time of locomotor activities and this is in agreement with the previous reports. Increased enzyme activity during the night hours would result in more glucose for conversion into the haemolymph disaccharide trehalose, an energy source for all physical activities of insects, including flight (Thompson, 2003). Trehalose concentration in the haemolymph of the cricket,Achetadomestica, was at peak at about 5:00 (3 h before dawn) (Nowosielski and Patton, 1964). Previous studies have shown that enzymatic assays performed on wholeCimexlectulariuslysates collected every 4 h under dusk and constant darkness conditions to determine the total GST, esterase and CYP450 activities revealed significant time-of-day specific changes and all the enzymes displayed the same peak activity during constant darkness (Khalidetal., 2019). Invertebrates are very active at night and become inactive during the daytime (Hodasi, 1982; Akinnusi, 2002). This could be associated with the nocturnal behaviour of insects as they perform their feeding activities during the night, thus making substrate available for the enzymes to act upon (Ademoluetal., 2018). Reports by Linetal., 2014 showed that the mRNA level of the delta-class glutathione S-transferase (BgGSTD1) ofBlattellagermanicadecreased early in the day and increased in the middle of the night, circadian fluctuation observed in the study coincides with its locomotor activity, reflecting the nocturnal nature of the cockroach (Lepplaetal., 1989; Lin and Lee, 1996). This study also showed that femoral muscle had significantly higher enzyme activities than thoracic muscle, this could be attributed to the fact that these insects are known to do more of walking and running than flying (Williametal., 2007).

In conclusion, our study revealed that the enzyme activities, and the contents of the organic and inorganic substances in the femoral and thoracic muscles of the 8-day-old adults ofP.americanavaried at various time points studied could be an indication that these time points were their peak period of enzyme and metabolic activities in the femoral and thoracic muscles, or it might be that the food substances that fuel the locomotor activities ofP.americanaadults are mobilized at those time points of the day. Secondly, our findings showed that these insects possess the ability to metabolize their organic and inorganic substrates with the help of the enzymes amylase, lipase, proteinase and α-glucosidase for movement at those various time points studied.