Arthropods, 2013, Vol. 2, Iss. 3

Arthropods

Vol. 2, No. 3, 1 September 2013

International Academy of Ecology and Environmental Sciences

Arthropods ISSN 2224-4255 Volume 2, Number 3, 1 September 2013 Editor-in-Chief WenJun Zhang Sun Yat-sen University, China International Academy of Ecology and Environmental Sciences, Hong Kong E-mail: [email protected], [email protected] Editorial Board Andre Bianconi (Sao Paulo State University (Unesp), Brazil) Anton Brancelj (National Institute of Biology, Slovenia) Hans-Uwe Dahms (Sangmyung University, Korea) A. K. Dhawan (Punjab Agricultural University, India) John A. Fornshell (Northern Virginia Community College, USA) Xin Li (Northwest A&F University, China) Oscar E. Liburd (University of Florida, USA) Ivana Karanovic (Hanyang University, Korea) Enoch A Osekre (KN University of Science and Technology, Ghana) Rajinder Peshin (Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, India) Michael Stout (Louisiana State University Agricultural Center, USA) Eugeny S. Sugonyaev (Russian Academy of Sciences, Russia)

Editorial Office: [email protected] Publisher: International Academy of Ecology and Environmental Sciences Address: Flat C, 23/F, Lucky Plaza, 315-321 Lockhart Road, Wanchai, Hong Kong Tel: 00852-6555 7188 Fax: 00852-3177 9906 Website: http://www.iaees.org/ E-mail: [email protected]

Arthropods, 2013, 2(3): 95-104

IAEES www.iaees.org

Article

The locomotory rhythmic activity in scorpions: with a review

Michael R. Warburg

Dept. of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel

E-mail: [email protected]

Received 21 February 2013; Accepted 25 March 2013; Published online 1 September 2013

Abstract

Locomotory rhythmic behavior is entrained by the change between photophase and skotophase and to some

extent by thermal conditions. In many species studied most activity takes place during early night hours. Some

species show completely a nocturnal activity pattern, whereas a few species are entirely diurnal. There does

not appear to be a pattern related to the timing and extent of the photophase. Except perhaps for Leiurus

quinquestriatus (Hemprich and Ehrenberg, 1829) which appears to be less active at the highest temperature.

This subject was studied in 30 species of scorpions most of them buthids (53.3%), that were studied so far in

42 different studies.

Keywords Scorpiones; diel locomotory rhythm; thermal ‘Zeitgeber’.

1 Introduction

The behaviour of scorpions has received less attention than it perhaps should have (Warburg and Polis, 1990).

Most cited studies were on rhythmic activity of scorpions. Here studies were divided between those on the

optic ‘Zeitgeber’ or time giver that bring about rhythmic activity (Warburg, in preparation). However most

studies were conducted on the behaviour of locomotory rhythmic activity. The activity patterns described here

are entrained by ambient conditions largely photophase and skotophase and to a certain extent also by thermal

factors. Such entrainment factor or ‘Zeitgeber’ is the factor that brings about this rhythmic activity (Cloudsley-

Thompson, 1961, 1978).

Dube and Fleissner (1985) describe three types of movements while in the wheel-running apparatus: (1)

slow pace movements typical of a circadian rhythms of 12L/12D light regime; (2) long-lasting runs, and (3)

rapid locomotion.

It was already Wuttke (1966) who described the bimodal activity in Euscorpius carpathicus (Linnaeus,

1767). In some species no rhythmic activity was noticeable. Thus, Cloudsley-Thompson and Constantinou

(1985) studying Opisthacanthus sp which did not show any rhythm. Cloudsley-Thompson (1973) found in

Buthotus minax (Koch, 1875) that the circadian locomotory rhythm is entrained by regular transition from dark

Arthropods ISSN 22244255 URL: http://www.iaees.org/publications/journals/arthropods/onlineversion.asp RSS: http://www.iaees.org/publications/journals/arthropods/rss.xml Email: [email protected] EditorinChief: WenJun Zhang Publisher: International Academy of Ecology and Environmental Sciences

Arthropods, 2013, 2(3): 95-104

IAEES www.iaees.org

to light and by rising temperatures. A similar situation was seen in Babycurus centrurimorphus (Karsch, 1886)

by Cloudsley-Thompson (1975) who studied this buthid and found that diurnal rhythm was entrained by shifts

both from dark to light and changes in temperatures.

Many of these studies show that activity is highest during the first few hours of the night (Cloudsley-

Thompson, 1978, 1980) whereas others show activity peaking at night. Thus, Cloudsley-Thompson found a

peak of activity in Scorpio maurus, Buthus occitanus (Amoreux, 1789) and Androctonus australis (Linnaeus,

1758) during the hours of 1800-2000 PM. The latter species was largely nocturnal (Constantinou, 1980). In

(1978) Cloudsley-Thompson (Tab. 1 therein) summarized the situation in the scorpion species studied up till

then. El Bakary and Fuzeau-Braesch (1988) studied: Leiurus quinquestriatus (Hemprich and Ehrenberg, 1829)

using three methods of detecting locomotory activity. They too found bimodal onset of activity coincides with

onset of photophase and skotophases.

Benton (1992) studying Euscorpius flavicaudis (DeGeer, 1778), found them active at dusk and dawn. In

this study I shall review the subject of locomotory rhythms in scorpions. This would include only the rhythmic

activities involving locomotion and not any rhythmic physiological functions. These were discussed recently

(Warburg, 2013). This subject was studied in 30 species of scorpions that were studied so far in 42 different

studies, listed as the follows:

Buthidae (16 species): 1. Androctonus australis (Linnaeus, 1758) Constantinou (1980); Baz et al. (2009) 2. Anomalobuthus rickmersi (Kraepelin, 1900) Fet (1980) 3. Babycurus centrurimorphus Karsch, 1886 Cloudsley-Thompson (1975) 4. Buthus hottentotta Fabricius, 1787 Toye (1970) 5. Buthus occitanus (Amoreux, 1789) Constantinou (1980); Constantinou & Cloudsley-Thompson (1980) 6. Buthotus occitanus (Amoreux, 1789) Skutelsky (1996) 7. Buthotus minax (L. Koch, 1875) Cloudsley-Thompson (1963, 1973) 8. Centruroides sculpturatus Ewing, 1928 Hadley & Williams (1968); Crawford & Krehoff (1975) 9. Hottentotta judaicus (E. Simon, 1872) Warburg & Ben-Horin (1979) 10. Leiurus quinquestriatus (Hemprich & Ehrenberg, 1829) Cloudsley-Thompson (1963); Abushama (1963); El Bakary & Fuzeau-Braesch (1988); Warburg & Ben-Horin (1979) 11. Liobuthus kessleri (Birula, 1898) Fet (1980) 12. Mesobuthus gibbosus (Brullé, 1832) Kaltsas & Mylonas (2010) 13. Mesobuthus eupeus (C. Koch, 1813) Fet (1980) 14. Mesobuthus caucasicus (Nordmann, 1840) Fet (1980) 15. Orthochirus scrobilosus (Grube, 1873) Fet (1980) 16. Parabuthus villous (Peters, 1862) Harrington (1981)

Euscorpiidae (2 species):

96

Arthropods, 2013, 2(3):

IAEES www.iaees.org

17. Euscorpius carpathicus (Linnaeus, 1767) Wuttke (1966) 18. Euscorpius flavicaudis (DeGeer, 1778) Cloudsley-Thompson & Constantinou (1983); Benton (1992) Cloudsley-Thompson (1963)

Hemiscorpiidae (1 species): 19. Hadogenes bicolor (Purcell, 1899) Constantinou (1980); Constantinou & Cloudsley-Thompson (1980)

Vaejovidae (3 species): 20. Paruroctonus boreus (Girard, 1854) Tourtlotte (1974) 21. Vaejovis mesaensis probably Paruroctonus mesaensis now Smeringurus mesaensis (Stahnke, 1957) Hadley & Williams (1968); Polis (1980) 22. Vaejovis confusus Stahnke, 1940 Hadley & Williams (1968)

Scorpionidae (8 species): 23. Diplocentrus spitzeri. Stahnke, 1970 Crawford & Krehoff (1975) 24. Nebo hierichonticus (Simon, 1872) Warburg & Ben-Horin (1979) 25. Pandinus gregoryi (Pocock, 1896) Constantinou (1980); Constantinou & Cloudsley-Thompson (1980) 26. Pandinus exitialis (Pocock, 1888) Cloudsley-Thompson (1963) 27. Pandinus imperator (C.L. Koch, 1841) Toye (1970) 28. Scorpio maurus fuscus Hemprich & Ehrenberg, 1829 Warburg & Ben-Horin (1979) 29. Heterometrus swammerdami (E. Simon, 1872) Cloudsley-Thompson (1981) 30. Heterometrus fulvipes (C.L. Koch, 1838) Babu, Reddy & Kasaiah (1988)

2 Materials and Methods

Measuring activity in scorpions was carried out largely by actograph use. The actograph used here consists of a

Perspex box measuring 5 x 6 x 20cm that rotated at its mid-point on a pivot. At one side an electrical contact

closed a circuit thereby signaling a change in the position of the actograph that resulted from the scorpion

moving inside the box. These movements were recorded on a Model 712 Telrad Recorder during the 24h

experiment. This actograph apparatus was placed into a Struers Refritherm where temperature was controlled

at ±0.50C. The animals could be observed through a window in the Refritherm’s door (see Warburg and Ben-

Horin, 1979).

The scorpion species studied here were collected from the field. Four scorpion species were used here:

Scorpio maurus fuscus (Hemprich and Ehrenberg, 1829), Nebo hierichonticus (Simon, 1872) Hotenttotta

judaicus (Simon, 1872) and L. quinquestriatus.

3 Results

Diel Activity in three scorpion species is given as percentage of time spent in activity during four-six hour

watches (Fig. 1). In all three species studied here most activity (between 55.3-87.6%) was spent active during

the 1st watch (06 PM- 12). The three scorpion species differed in the amount of time spent during the

97

Arthropods, 2013, 2(3): 95-104

IAEES www.iaees.org

remainder of the day. Thus, whereas N. hierichonticus and H. judaicus had shown hardly any activity (12.3%

and 21.8% respectively), S. m. fuscus was active also during the remaining of the day (44.8%).

Fig. 1 Diel activity (%) in three scorpion species.

The number of activity runs at four temperatures (200C, 250C, 280C, 350C) was measured in four scorpion

species (Fig. 2). These scorpion species differed in their response in a temperature gradient. Thus, the peak of

activity was at the lowest temperature at 200C in L. quinquestriatus (55), and in N. hierichonticus (40.6),

whereas for S. m. fuscus and H. judaicus it was at 250C (69.1 and 41 respectively). At the highest temperature

tested here (350C) the two buthid species (L. quinquestriatus and H. judaicus) have shown the lowest activity

(5.5 and 16.3 respectively).

When the number of activity runs was compared in illuminated and dark parts of the thermo-preferendum

apparatus (Fig. 3), outstanding differences in the activity of the four scorpion species were noticeable. Thus in

N. hierichonticus and S. m. fuscus there was no marked difference between the activity in the dark and

illuminated parts of the thermo-preferendum apparatus. In the two species activity was higher in the

illuminated part. In the first species it was 29.6 at 200C, whereas in the latter species it was 29.6 at 250C. In the

two buthids activity was higher in the darkened part. Thus in H. judaicus it was highest at 250C (32.5) whereas

in L. quinquestriatus it peaked at 280C (34.4).

98


IAEES www.iaees.org

Fig. 2 Average number of activity runs at four temperatures in four scorpion species.

Lastly, the time spent in the thermo-preferendum apparatus was examined in detail in 300C thermal zones

ranging between 150C–300C (Fig. 4). This was examined in S.m. fuscus and N. hierichonticus. Both species

have shown similar results with activity rising between 210C-270C (27.8-37.3 in the first species, and 30-36.7

in the second species), dropping thereafter between 270C-300C (to 23.3 in the first species, and 8.7 in the latter).

4 Discussion

4.1 Activity during daytime

Constantinou (1980) studied four scorpion species: Pandinus gregoryi (Pocock, 1896) showed diurnal activity

most of its time (63%), Hadogenes bicolor (Purcell, 1899) with 26% activity diurnal, A. australis was largely

nocturnal, as was also B. occitanus. Constantinou and Cloudsley-Thompson (1980) studied four different

scorpion species: Scorpio maurus, B. occitanus. P. gregoryi and H. bicolor. They found the last two are

largely diurnal as was the case also with Parabuthus villosus (Peters, 1862) where Harrington (1981) found it

to be diurnal.

55

36.139.4

5.5

0

10

20

30

40

50

60

20 25 28 35

Av. n

o. ru

ns

Temp. (C)

L. quinquestriatus

20

41

24.3

16.3

05

1015202530354045

20 25 28 35

Av. n

o. ru

ns

Temp. (C)

H. judaicus

40.6

24.6

13

28.5

05

1015202530354045

20 25 28 35

Av. n

o. ru

ns

Temp. (C)

N. hierichonticus

39.8

69.1

56.9

30

01020304050607080

20 25 28 35

Av. n

o. ru

ns

Temp. (C)

S. m. fuscus

99

Arthropods, 2013, 2(3): 95-104

IAEES www.iaees.org

Fig. 3 Average number of activity runs in illuminated (grey bars) and dark arenas.

4.2 Activity during early evening

Hadley and Williams (1968) studied three species of scorpions: Vaejovis confusus (Stahnke, 1940), V.

mesaensis (Stahnke, 1957) and Centruroides sculpturatus (Ewing, 1928). All of them showed peak activity in

the evening. Similarly, Toye (1970) studying both Pandinus imperator (Koch, 1841) and Buthus hottentotta

Fabricius, used an actograph made of celluloid tubes pivoting through transverse center. He too found that the

29

9.45

1.5

26 26.7

34.4

4

05

10152025303540

20 25 28 35

Av

. n

o.

acti

vit

y ru

ns

Temp. (C)

L. quinquestriatus

4

8.5 8.66

16

32.5

15.7

10.3

0

5

10

15

20

25

30

35

20 25 28 35

Av

. n

o. o

f ac

tiv

ity

run

s

Temp. (C)

H. judaicus

29.6

13.6 1511 11

13 13.5

0

5

10

15

20

25

30

35

20 25 28 35

Av

. n

o.

of

acti

vit

y ru

ns

Temp. (C)

N. hierichonticus

22.4

29.6

4

29

17.4

11

16

26

0

5

10

15

20

25

30

35

20 25 28 35

Av

. n

o. a

ctiv

ity

run

s

Temp. (C)

S. m. fuscus

100


IAEES www.iaees.org

peak activity was between 1500-1800h in both species. Polis (1980) studying the vaejovid Paruroctonus

mesaensis (Stahnke), found that surface activity increases during early evening decrease after 1400AM.

Warburg and Ben-Horin (1979) studied effect of temperature on rhythm of three scorpion species in an

actograph. They found in S. m. fuscus high activity 1200-1800h and 1800-2400h. This activity dropped

drastically with rising temps (to 280C and 350C). N. hierichonticus showed high activity between 1800-2400hr

during 06-12 hrs, whereas H. judaicus rhythm was not affected by temperature. Babu, Reddy and Kasaiah

(1988) studied Heterometrus fulvipes (Koch, 1838) with an actograph.

Fig. 4 Percentage time spent in the thermo-preferendum apparatus.

S.m. fuscus

2023.3

47.5

2.56.7

0

10

20

30

40

50

18-21 21-24 24-27 27-30 30-34

Temp. ranges (C)

Tim

e s

pe

nt

(%)

N.hierichopnticus

51.7

43.3

4.20.8

0

10

20

30

40

50

60

18-21 21-24 24-27 27-30 30-34

Temp. ranges (C)

Tim

e s

pe

nt

(%)

L.quinquestriatus

21.7

0.8

52.5

25

0

10

20

30

40

50

60

18-21 21-24 24-27 27-30 30-34

Temp. ranges (C)

Tim

e s

pe

nt

(%)

101

Arthropods, 2013, 2(3): 95-104

IAEES www.iaees.org

4.3 Activity during night

Cloudsley-Thompson (1963) Studied three scorpion species: Pandinus exitialis (Pocock, 1888), L.

quinquestriatus and Buthotus minax (see Fig. 2 therein). All of these species were nocturnal. One of these

scorpions L. quinquestriatus, was studied in an actograph apparatus box pivoted about its median transverse

axis writing on a barograph drum that acted as a kymograph (Abushama, 1963). He too found it to be

nocturnal following an endogenous factor of the rhythm.

On the other hand, Tourtlotte (1974) who studied surface activity of Paruroctonus boreus (Girard, 1854),

found its activity peaked at 2130h. Likewise, Crawford and Krehoff (1975) studying Centruroides sculpturatus

and Diplocentrus spitzeri (Stahnke, 1970), found that surface activity in the field peaked between 2030-2230h

whereas, activity in the actograph showed nocturnal activity. Only Centruroides spitzeri showed endogenous

circadian rhythm in constant darkness. Fet (1980) studied the buthids: Orthochirus scrobilosus (Grube, 1873),

Anomalobuthus rickmersi (Kraepelin, 1900), Mesobuthus eupeus (Koch, 1813), M. caucasicus (Nordmann

1840) and Liobuthus kessleri (Birula 1898). In all these species activity peaked between 2100-0100 h.

Cloudsley-Thompson (1981) studied Heterometrus swammerdami (Simon, 1872) and found it active

especially at night. Cloudsley-Thompson and Constantinou (1983) studying E. flavicaudis described their

nocturnal habits in the field and during actograph studies.

Baz, Sallam and El-Naggar (2009) studied A. australis locomotor activity rhythm. It was synchronized

with electroretinogram rhythm starting at night. Skutelsky (1996) studying: Buthotus occitanus israelis, found

its activity dropped with full moon. Kaltsas and Mylonas (2010) studying Meobuthus gibbosus found their

activity dropped with moonlight.

There are several points that emerge from this review:

1. Most of these studies (54%) were conducted on buthids that are usually the more active species.

2. Many of the studies did not remark anything about the ecology or physiological conditions of the scorpions

studied. This is especially important since in many studies the scorpions were previously kept for sometime

before the onset of the experiment.

3 The findings of these studies are not conclusive. Under what condition does a shift in behaviour of the

scorpion's rhythmic activity take place?

Acknowledgements

The assistance of A. Ben-Horin during his M.Sc. thesis work is gratefully acknowledged. The author is

indebted to Prof. Cloudsley-Thompson for providing an outlet to early publications, and to Dr. Polis for

inviting me to write a chapter in a book he was editing.

References

Abushama EF. 1963. Bioclimate, diurnal rhythms and water-loss in the scorpion, Leiurus quinquestriatus (H

& E). Entomological Monthly Magazine, 98: 216-224

Babu K.S, Reddy TG, Kasaiah A. 1988. Daily patterns of locomotor activity in the Indian scorpion

Heterometrus fulvipes (C.L. Koch). Indian Journal of Experimental Biology, 26: 307-311

Baz E-S, Sallam A E-D, El-Naggar MHR. 2009. Development of method for monitoring the locomotor and

visual circadian rhythms in a free-moving scorpion. Egyptian Journal of Natural Toxins, 6(2): 16-30

Benton TG. 1992. The ecology of the scorpion Euscorpius flavicaudis in England. Journal of Zoology

(London), 226: 351-368

Cloudsley-Thompson JL. 1956. Studies in diurnal rhythms – VI. Bioclimatic observations in Tunisia and their

102


IAEES www.iaees.org

significance to the physiology of the fauna, especially woodlice, centipedes, scorpions and beetles. Annals

and Magazine of Natural History, 12(9): 305-329

Cloudsley-Thompson JL. 1961. Observations on the biology of the scorpion Leiurus quinquestriatus (H. & E.)

in the Sudan. Entomological Monthly Magazine, 97: 153-155

Cloudsley-Thompson JL. 1963. Some aspects of the physiology of Buthotus minax (Scorpiones: Buthidae)

with remarks on other African scorpions. Entomological Monthly Magazine, 98: 243-246

Cloudsley-Thompson JL. 1973. Entrainment of the "circadian clock" in Buthotus minax (Scorpiones:

Buthidae). Journal of Intedisciplinary Cycle Research, 4(2): 119-123

Cloudsley-Thompson JL. 1975. Entrainment of "circadian clock" in Babycurus centrurimorphus (Scorpiones:

Buthidae). Journal of Intedisciplinary Cycle Research, 6: 185-188

Cloudsley-Thompson JL. 1978. Biological clocks in Arachnida. Bulletin of the British Arachnological Society,

4(4): 184-191

Cloudsley-Thompson JL. 1981. A comparison of rhythmic locomotory activity in tropical forest Arthropoda

with that in desert species. Journal of Arid Environments, 4: 327-334

Cloudsley-Thompson JL, Constantinou C. 1983. How does the scorpion Euscorpius flavicaudis.(Deg.) manage

to survive in Britain? International Journal of Biometeorology, 27(2): 87-92

Cloudsley-Thompson JL, Constantinou C. 1985. The circadian rhythm of locomotory activity in a Neotropical

forest scorpion, Opisthacanthus sp. (Scorpionidae). International Journal of Biometeorology, 29(1): 87-89

Constantinou C. 1980. Entrainment of the circadian rhythm of activity in desert and forest inhabiting scorpions.

Journal of Arid Environments, 3: 133-139

Constantinou C, Cloudsley-Thompson JL. 1980. Circadian rhythms in scorpions. 8th Internationaler

Arachnologen Congress Wien, 53-55

Crawford CS, Krehoff RC. 1975. Diel activity in sympatric populations of the scorpions Centuroides

sculpturatus (Buthidae) and Diplocentrus spirtzeri (Diplocerntridae). Journal of Arachnology, 2: 195-204

Dube C, Fleissner G. 1985. Temporal patterns of wheel-running behaviour in the scorpion Androctonus

australis. Journal of Intedisciplinary Cycle Research, 16(4): 250-251

El- Bakary Z, Fuzeau-Braesch S. 1988. Circadian rhythms and time measurement in locomotor activity of the

scorpion Leiurus quinquestriatus (Buthidae). Chronobiology Internationl, 5(2): 167-174

Fet VY. 1980. Ecology of the scorpions (Arachnida, Scorpiones) of the southeastern Kara-Kum.

Entomologoscheskoe Obozrenie, 59: 167-170

Hadley NF, Williams SC. 1968. Surface activities of some North American scorpions in relation to feeding.

Ecology, 49(4): 726-734

Harrington A. 1981. Diurnalism in Parabuthus villosus (Peters)(Scorpiones, Buthidae). Journal of

Arachnology, 10: 85-86

Kaltsas D, Mylonas M. 2010. Locomotory activity and orientation of Mesobuthus gibbosus (Scorpiones:

Buthidae) in central Aegean Archipelago. Journal of Natural History, 44(23-24): 1445-1459

Polis GA. 1980. Seasonal patterns and age-specific variation in the surface activity of a population of desert

scorpions in relation to environmental factors. Journal of Animal Ecology, 49: 1-18

Ramakrishna T, Rao KP. 1971. State determined system of a circadian rhythm in scorpion. Proceedings of the

Indian Academy of Science Section B, 73(4): 202-207

Skutelsky O. 1996. Predation risk and state-dependent foraging in scorpions: effect of moonlight on foraging

in the scorpion Buthus occitanus. Animal Behaviour, 52: 49-57

Tourtlotte GI. 1974. Studies on the biology and ecology of the northern scorpion, Paruroctyonus boreus

(Girard). Great Basin Naturalist, 34(3): 167-179

103

Arthropods, 2013, 2(3): 95-104

IAEES www.iaees.org

Toye SA. 1970. Some aspects of the biology of two common species of Nigerian scorpions. Journal of

Zoology London, 162: 1-9

Warburg MR, Ben-Horin A. 1979. Thermal effect on the diel activity rhythm of scorpions from mesic and

xeric habitats. Journal of Arid Environments, 2: 339-346

Warburg MR, Polis GA. 1990. Behavioral responses, rhythms and activity patterns. In: The Biology of

Scorpions (Polis GA, ed). 224-246, Stanford University Press, Stanford, USA

Warburg MR. 2013. A review on the diel rhythmic activities in physiological functions of scorpions.

Entomological Science, 16(3): 278-283

Wuttke W. 1966. Untersuchungen zur Aktivitätsperiodik bei Euscorpius carpathicus. Zeitschrift der

Vergleichende Physiologie, 53: 405-448

104

Arthropods, 2013, 2(3): 105-110

IAEES www.iaees.org

Article

Two records of Macrophthalmus Desmarest, 1823 (Decapoda:

Brachyura: Thoracotremata) from the NW of the Arabian Gulf

Amaal Gh. Yasser, Ibtisam M. AbdulSahib, Murtada D. Naser, Khalid Kh. S. Al-Khafaji, Haider Sh. Darweesh Department of Marine Biology, Marine Science Centre, University of Basrah, Basrah, Iraq


Received 1 March 2013; Accepted 5 April 2013; Published online 1 September 2013

Abstract

Specimens of two crabs Macrophthalmus dentipes Lucas, 1836 and Macrophthalmus laevis A. Milne-Edwards,

1867 were collected from the intertidal zone of the lower reaches of Shatt Al-Arab at Fao region, Basrah, Iraq,

2012. A note on the morphological features of these two species and a photograph is provided to confirm the

identification of the crabs.

Keywords Macrophthalmus dentipes; Macrophthalmus laevis; Brachyura; Shatt Al-Arab; Arabian Gulf.

1 Introduction

The family Macrophthalmidae Dana, 1851, is represented by two subfamilies Ilyograpsinae Števcic, 2005 and

Macrophthalminae Dana, 1851 in the Arabian Gulf. The first one subfamily Ilyograpsinae Števcic, 2005 is

represented by one species Ilyograpsus rhizophorae, While the second subfamily Macrophthalminae Dana,

1851 is represented by eight species, have been recorded from the area, all belonging to Macrophthalmus

Desmarest, 1823, represented of these: M. sinuspersici Naderloo & Türkay, 2010, Macrophthalmus graeffei

A. Milne-Edwards, 1873, M. dentipes Lucas, 1836, M. depressus Rüppell, 1830, M. grandidieri A. Milne-

Edwards, 1867, M. laevis A. Milne-Edwards, 1867, M. serenei Takeda & Komai, 1991, and M. sulcatus H.

Milne-Edwards, 1852, are important elements of the intertidal soft bottom communities (Naderloo et al., 2011).

Barnes (1970) was so far recorded M. dentipes from the Arabian Gulf at Al-Faw referred to it as M.

pectinipes Guerin-Méneville 1838. Pretzmann (1971) and Jones (1986) recorded the species from Bandar-

Abbas at the Iranian coast and from Kuwait respectively, both following Barnes (1970) using the name M.

pectinipes. Holthuis (1995) revised M. dentipes as the valid name by showing that M. pectinipes is an objective

synonym of M. dentipes, and therefore must be replaced by the latter. Apel and Türkay (1999) and Apel (2001)

listed M. dentipes from the Arabian Gulf with reference to the records by Barnes (1971) and Jones (1986).


Arthropods, 2013, 2(3): 105-110

IAEES www.iaees.org

Pretzmann (1971) described M. ressli as a new species from Bandar-Abbas, on the Iranian coast near the

Straits of Hormuz. Barnes (1976) synonymised M. ressli with M. laevis and was the first to provide a detailed

description for M. laevis.

The aim of the present study is to re-describe M. dentipes from fresh specimens collected from NW of the

Arabian Gulf at Faw region and to record Macrophthalmus laevis to add to the brachyua list of Iraq.


Specimens of M. dentipes and M. laevis were recently collected from the intertidal muddy flats of NW of the

Arabian Gulf, Fao region (Fig. 1) on July 2012. Specimens are preserved in 70% alcohol and deposited in the

marine science centre (MSC) (collection number: 33 and 34), and M. dentipes only deposited in the Zoological

Reference Collection of the Raffles Museum of Biodiversity Research, National University of Singapore

(ZRC).

The main abiotic parameters in the study area by the time of collection were as follows: salinity 35 ppt,

water temperature 27 oC, pH 8.4.

Fig. 1 Sampling site Faw=Fao region (white dot).

3 Results and Remarks

Macrophthalmus dentipes Lucas, 1836

Systematics

Order Decapoda

Macrophthalmidae Dana, 1851

Subfamily Macrophthalminae Dana, 1851

Macrophthalmus dentipes Lucas, 1836

(Fig. 2A, B, C and D)

Macrophthalmus dentipes Lucas 1836: 551. — Holthuis 1995: 401.

Macrophthalmus pectinipes — Guerin-Méneville 1838: 1, pl. 23. — Alcock 1900: 377. — Chhapgar 1957b:

106

Arthropods, 2013, 2(3): 105-110

IAEES www.iaees.org

512. — Barnes

1970: 237, fig. 10. — Pretzmann, 1971: 31; 1974: 442. — Tirmizi 1981: 109. — Titgen 1982: 253 (in list). —

Jones 1986:

159, pl. 45. — Tirmizi & Ghani 1996: 121, fig. 46.

Macrophthalmus (Venitus) dentipes — Apel & Türkay 1999: 135. — Apel 2001: 110. — Naderloo et al.,

2011: figs. 4a–e, 5a–f, 10a–b.

Type locality

Bombay (= Mumbay), India

Material examined (msc, 33)

Carapace measurements are length × breadth respectively.

Three (38.55×60.50), (36.55×60.00), (39.00×60.50) mm collected during July 2012 from the intertidal

zones of the mudflats of lower reaches of Shatt Al-Arab at Fao.

Diagnosis

Carapace (Fig. 2 A) moderately wider than long (CB/CL = 1.6), slightly convex; large granules scattered on

entire posterior surface except in narrow median, frontal regions. Lateral margin of carapace (Fig. 2 A) with 3

distinct teeth (including exorbital tooth); first nearly subquadrate, with posterior margin smooth, curved

forward; second triangular, with smooth margin, higher than first, greatest width of carapace between second

lateral teeth; third very small, directed forwards; posterolateral margin nearly straight, slightly converging

posteriorly, with small granules, beset with long setae; posterior margin with very small granules.

Eyestalks narrow (Fig. 2A), long, but not reaching to exorbital angle.

Male abdomen (Fig. 2B) with segments 3, 4 of same length, slightly shorter than segment 5; segments 5, 6

nearly of same length, with lateral margins nearly straight; lateral margins of segment 6 with small depression

at one third distal portion; telson very slightly longer than segment 6, lateral margins strongly converging

distally, apically rounded.

Palm long (Fig. 2C), outer surface smooth without longitudinal ridge; inner surface smooth, patch of dense

setae on upper portion. Fingers remarkably curved inward distally, movable finger with upper margin smooth,

long setae densely along inner surface of upper margin, continuous on upper, outer surface, cutting edge with

subproximal differentiated tooth, small teeth distally; immovable finger narrow, with relatively large teeth on

cutting edge, long setae along inner surface.

Male G1 (Fig. 2D,E,F) moderately stout, slightly curved outward medially; distal half relatively narrowing,

with apical chitinous process remarkably long, narrow, curved outward at about 45°; distal opening large,

distinct, subdistal on dorsal portion of apical process; long feather-shaped setae densely set along lateral

margin, long setae around apical process.

Habitat

Macrophthalmus dentipes is the largest ocypodid crab, inhabiting the mid and low intertidal zones in muddy

sand/sandy mud substrates at the lower reaches of Shatt Al-Arab at Fao, it may be occur coexisting with the

grapsoid crab Metaplax indica. Macrophthalmus dentipes digs large burrows with an opening of

approximately 10 cm in diameter.

Distribution

Northern Indian Ocean: northern and eastern Arabian Gulf, Oman (Gulf of Masirah), Pakistan, west coast of

India, Iraq.

Macrophthalmus laevis A. Milne-Edwards, 1867

Systematics

107

Arthropods, 2013, 2(3): 105-110

IAEES www.iaees.org

Order Decapoda

Macrophthalmidae Dana, 1851

Subfamily Macrophthalminae Dana, 1851

Macrophthalmus laevis A. Milne-Edwards, 1867

(Fig3. A,B,C and D)

Macrophthalmus laevis A. Milne-Edwards 1867: 287. — Barnes 1976: 143, fig. 6a–c. — Titgen 1982: 150.

Macrophthalmus (Macrophthalmus) ressli Pretzmann 1971: 382, pl. 9 figs. 23.

Macrophthalmus resseli [sic!] — Pretzmann,1974: 441.

Macrophthalmus (Macrophthalmus) laevis — Barnes 1977: 277 (in key), 280 (in list); 2010: 35 (in key), 40.

— Tirmizi & Ghani 1988: 253, figs. 1–11. — Tirmizi & Ghani 1996: 109, fig. 41. — Apel & Türkay 1999:

135. — Apel 2001: 109— Naderloo et al., 2011: figs. 13a–f, 14a–e, 10e–f.

Type locality

Indian Seas.

Material examined (msc,34)

Carapace measurements are length × breadth respectively.

Two males (12.50×24.50) and (12×23.50) mm collected during July 2012 from the intertidal zones of the

mudflats of lower reaches of Shatt Al-Arab at Fao.

Carapace

Macrophthalmus laevis is a medium-size species (Fig. 3A) wider than long; posterior surface is convex, small

granules distributed on posterior surface, extensive patch of setae near posterolateral margin, long setae on

lateral margin. Regions well defined; defining gastric, epibranchial regions are remarkably deep. Lateral

margin with three teeth including exorbital angle.

Chelipeds nearly equal; merus with upper surface smooth, inner, upper margins sparsely beset with long

setae, row of long setae near inner margin.Carpus smooth with large spine- shaped tooth medially on upper

inner margin, small one behind it, two spine-shaped teeth on inner proximal margin. Movable finger long ( Fig.

3C) curved inward distally; upper margin smooth; cutting edge with differentiated subproximal tooth, large,

subquadrate, low, small denticles distal to large one along cutting edge. Immovable finger short, with median

tooth, large, extending proximally, small denticles on cutting edge, even on large tooth.

Walking legs narrow, long, anterior margin of segments bearing long setae. Merus with small subdistal

tooth on anterior margin, that of second, third legs large, last leg usually lacking this subdistal tooth ( Fig.3.A).

Male abdomen (Fig. 3B) triangular; segments 3, 4 of same length, segment 5 slightly longer; segment 6

longest with lateral margins swollen proximally, gently converging distally; telson slightly shorter than

segment 6, with margins clearly converging distally, rounded distally.

Male G1 (Fig. 3D) curved outward medially; apical chitinous process short, nearly subdistal, directed

laterally at 45°; distal opening prominent, located apically; long setae around apical part, long plumose setae

sparsely set along lateral, ventral surfaces.

Habitat

M. laevis mainly in the upper mid littoral zone on muddy silty substrata.

Distribution

North-western Indian Ocean: Persian Gulf, Gulf of Oman, Pakistan, Iraq.

108

Arthropods, 2013, 2(3): 105-110

IAEES www.iaees.org

Fig. 2 Macrophthalmus dentipes Lucas,1836, male (39.00×60.50): A, posterior view of whole crab, male; B, male ventral view. C, cheliped of male, outer surface; D- F, first gonopod, Photos taken by Murtada.D.Naser, Marine Science Centre.

Fig. 3 Macrophthalmus laevis A. Milne-Edwards 1867, male (12.5×24.5): A, posterior view of whole crab, male; B, male ventral view. C, cheliped of male, outer surface; D,first gonopod, Photos taken by Murtada.D.Naser, Marine Science Centre.

109

Arthropods, 2013, 2(3): 105-110

IAEES www.iaees.org

Acknowledgements

M.D.N. thanks Dr Peter K.L. Ng (Raffles Museum of Biodiversity Research, Department of Biological

Sciences, National University of Singapore) for confirming the identity of Macrophthalmus dentipes.

References

Alcock A. 1900. Material for a carcinological fauna of India. No. 6: The Brachyura Catometopa, or

Grapsoidea. Journal of the Asiatic Society of Bengal, 69 (2): 279-456

Apel M. 2001. Taxonomie und Zoogeographie der Brachyura, Paguridea und Porcellanidae (Crustacea:

Decapoda) des Persisch Arabischen Golfes unpublished PhD. Thesis. Johann Wolfgang Goethe-Universität,

Frankfurt am Main, Germany

Apel M, Türkay M. 1999. Taxonomic composition, distribution and zoogeographic relationships of the grapsid

and ocypodid crab fauna of intertidal soft bottoms in the Arabian Gulf. Estuarine, Coastal and Shelf

Science, 49(Suppl. A): 131-142

Al-Zaidan ASY, Kennedy H, Jones DA, et al. 2004. Role of microbial mats in Sulaibikhat Bay (Kuwait)

mudflat food webs: evidence from δ13C analysis. Marine Ecology Progress Series, 38: 27-36

Barnes RSK 1970. The species of Macrophthalmus in the collections of the British Museum (Natural History).

Bulletin of the British Museum of Natural History, 20: 203-251

Barnes RSK 1976. Contributions towards a revision of Macrophthalmus, VIII: A re-examination of the M.

telescopicus Owen complex; the status of M. laevis H. Milne-Edwards; and the affinities of M. holthuisi

Sérene. Zoologische Mededelingen, 50(10): 133-151

Barnes RSK 1977. Concluding contribution towards revision of, and a key to, the genus Macrophthalmus

(Crustacea Brachyura). Journal of Zoology London, 182: 267-280

Holthuis LB 1995. The identities of Macrophthalmus rouxii Lucas, 1836, and M. dentipes Lucas, 1836, and

the substitution of the latter name for M. pectinipes Guérin, 1838 (Decapoda, Brachyura, Ocypodidae).

Crustaceana, 68(3): 401-403

Jones DA 1986. A Field Guide to the Sea Shores of Kuwait and the Arabian Gulf. University of Kuwait,

Blandford Press, Poole, Kuwait

Lucas H. 1836. Macrophthalme, Macrophthalmus. (Crust.) In: Guérin-Meneville, F.E.: Dictionaire pittoresque

d’Histoire naturelle Vol. 4, pp. 551, pl. 315, fig. 5.

Naderloo R, Türkay M, Apel M. 2011. Brachyuran crabs of the family Macrophthalmidae Dana, 1851

(Decapoda: Brachyura: Macrophthalmidae) of the Persian Gulf. Zootaxa, 2911: 1-42

Tirmizi NM, Ghani N. 1988. The rediscovery of Macrophthalmus (Macrophthalmus) laevis A. Milne-

Edwardds, 1867, in the Arabian Sea (Decapoda Brachyura). Crustaceana, 55(3): 253-256

Tirmizi NM, Ghani N. 1996. Marine Fauna of Pakistan: 5: Crustacea: Brachyura, Brachyrhyncha, Part 1:

Xanthidae Goneplacidae, Pinnotheridae, Ocypodidae, Grapsidae. Center of Excellence, University of

Karachi, Pakistan

Titgen RH. 1982. The Systematics and Ecology of the Decapods of Dubai, and their Zoogeographic

Relationships to the Arabian Gulf and the Western Indian Ocean. Unpublished D. Phil. Thesis, Texas A &

M University, USA

110

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

Article

Reproductive characteristics of a brachyuran crab, Grapsus

tenuicrustatus (Herbst, 1783) (Decapoda: Grapsidae) found in Talim

Bay, Batangas, Philippines

Michael A. Clores1, Gliceria B. Ramos2 1Ateneo de Naga University, Naga City, Philippines 2Biology Department, De La Salle University, Taft Avenue, Manila, Philippines


Received 7 March 2013; Accepted 10 April 2013; Published online 1 September 2013

Abstract

The study determined some reproductive characteristics of a brachyuran crab, Grapsus tenuicrustatus (Herbst,

1783), one among the most widespread and diverse groups of invertebrates. Results revealed that there were

more males (52.94%) than females (47.06%) collected at the study sites with a sex ratio of 1:1.13. Thirty

percent (30%) of the samples were ovigerous females. Ovigerous females have the largest caraface length, CL,

(31.25 ± 1.43) compared with the males (22.14 ± 0.726) and non-ovigerous females (26.63 ± 1.12). Based on

one-way ANOVA, the differences were significant. Difference between non-ovigerous and ovigerous females

was also found significant based on t-test for independent samples. There was a non-conspicuous bi-modal size

distribution for all the crabs, with non-normal distributions for all crabs and for males, but not when all

females or ovigerous females only were grouped together. The size-frequency distributions of males and

females are significantly different from each other similar with that observed between the size-frequency of

ovigerous and non-ovigerous females. There were more ovigerous crabs belonging to the first year age class

(CL = 16 - 33) (53.13 %) than those that belong to the older class (CL = 34 - 43) (46.88 %). Fecundity ranged

from 4400 (CL = 16 mm) to 26400 (CL = 43 mm) eggs. Egg volume ranged from 0.40 ml to 2.40 ml, egg

diameter from 1.1 µm to 5.0 µm with an average diameter of 3.170 µm and egg count from 4400 to 26400

with a mean of 12684 eggs. Egg number was positively correlated with female size.

Keywords Grapsus tenuicrustatus; brachyuran crab; fecundity; ovigerous; reproductive characteristics.

1 Introduction

In the Philippines, brachyurans (true crabs) are among the most widespread and diverse groups of invertebrates.

One of the interesting brachyurans are the Sally-lightfoot crab or natal sally-lightfoot or shore crab, Grapsus

tenuicrustatus, locally called “Katang”, because these are known to be harvested and processed by


Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

fermentation as a delicacy and crab roe fat.

A random search of previous studies on brachyurans in the Philippines showed a paucity of research on

their reproductive ecology and the environmental and biological constraints that influence them as a population.

Indeed, while crab population structure and reproduction of subtropical species have become major research

agenda (Spivak et al. 1991; Mouton and Felder 1995), such studies on the tropical ones are still needed (Litulo,

2005).

Understanding the breeding potential of many well-dispersed marine invertebrates, like crabs, entails

determining intraspecific variation of reproductive characteristics (Dugan, 1991). Incomplete knowledge of the

life history of the organisms that comprise communities is one of the principal problems in understanding how

marine ecosystems function. For crab populations, understanding the environment and biological constraints

that are shaping them (Oshiro, 1999; Litulo, 2005) remains one important aspect of marine ecology.

In most marine invertebrates, the newly laid eggs contain all the energy and reserves for embryonic

development (Holland, 1978; Jaeckle, 1995). In species with complex life cycles, larval survival and growth

may depend on the energy reserves that remain after hatching (Paschke, 1998; George, 1999). Consequently,

these depend on the initial egg reserves and their utilization during embryogenesis. The embryonic

development occurs in a variety of modes, e.g. free developing, encapsulated, incubated (Sastry, 1983; Levin

and Bridges, 1995), and under a particular combination of environmental factors that may affect the embryonic

energy budget and thus, larval reserves (Gimé́nez and Anger, 2001). For decapods, the marine benthos is the

typical environment. The larvae hatch from the eggs attached to the female’s abdominal appendages (pleopods)

and develop in the marine plankton for several weeks (Diesel, 1989).

The present study determined some reproductive characteristics of G. tenuicrustatus (Herbst, 1783).

Specifically, it described the following: (i) total number and sex ratios of collected crabs; (ii) the size and age

distribution of ovigerous crabs; (iii) the female size at maturity and age classes based on carapace length (CL,

mm) and their size-frequency distribution, and (iv) an estimate of population fecundity (e.g., volume, number,

and diameter of eggs; relative number of females carrying various egg stages; estimate number of age in

different size groups of ovigerous crabs, and the relationship between egg number and female size). Broadly,

results of this study could provide insights on the relative plasticity or conservation of different reproductive

characteristics and the effects of intraspecific variation in those characteristics on the reproductive potential of

a well-dispersed marine invertebrate (Dugan, 1991).


2.1 Study site

Samples of G. tenuicrustatus were collected in rocky areas near seagrass beds at Talim Point (130 57’ 55.43’’,

1200 36’ 20.36’’ E), a portion of Talim Bay, Barangay Ligtasin, Lian, Batangas, Philippines. Talin Bay is

located between latitude 130 58.8’ North and longitude 1200 38.0’ East of DLSU Br. Alfred Shields FSC

Marine Biology Station, approximately 200 km. south of Manila fronting South China Sea.

Laboratory activities were conducted at the Biology Laboratories of Ateneo de Naga University, Naga City,

Philippines.

2.2 Sampling method

G. tenuicrustatus were collected opportunistically with a shovel and by hand from aggregations of crabs in the

wash and surf zones of the bay following previous methods (Wenner et al., 1987; Dugan, 1990; Dugan et al.,

1991). The crabs were retained and separated from the sand by washing through mesh bags. A total number of

153 crabs were collected.

112

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

2.3 Female size and age

Size-frequency distributions were determined by individual measurement of carapace lengths (CL), to the

nearest millimeter, using a vernier caliper. The sex and reproductive condition of all crabs were recorded (i.e.,

ovigerous or non-ovigerous). The size-frequency distributions of female crabs were examined for modal

breaks. Since there were two non-overlapping size modes, the mode of larger ovigerous crabs were assigned to

the older overwintered age class (Years 2 and 3) and the mode of smaller ovigerous crabs were assigned to the

first year class (young of one year) following Dugan et al. (1991).

The size at maturity, and the minimum (5th percentile) and maximum (95th percentile) sizes of ovigerous

female crabs were determined from size-frequency distributions for each sample. The 5th and 95th percentile

sizes of ovigerous crabs were determined from the cumulative number of ovigerous crabs and were used as

estimates of the minimum and maximum sizes of ovigerous female crabs. For the whole sample, the

proportions of ovigerous crabs were calculated for three categories, as follows: (1) the proportion of ovigerous

crabs above the size of the smallest ovigerous crab; (2) the proportion of ovigerous crabs assigned to the older

age class; and (3) the proportion of ovigerous crabs assigned to the first year class.

The developmental stage of each clutch was determined using the method of Eickstaedt (1969), which

divides egg development into ten stages. Stages 1-4 were of most interest. The amount of cleavage and the

proportion of the egg that is free of orange yolk distinguish these stages. Stage l eggs are uncleared or in a state

of cleavage; in Stage 2 cleavage is complete; in Stage 3 up to I/4 of the egg is free of yolk, and in Stage 4 up to

1/3 of the egg is free of yolk. Stage 5 eggs have visible embryonic eye pigment and were not used in fecundity

estimates. Forty (40) eggs were randomly selected and their diameter was measured with an ocular micrometer.

Finally, descriptive statistics (e.g., mean, mode, median, standard error of the mean, minimum, maximum,

range, standard error of the sizes) of egg diameter were reported.

2.4 Population fecundity

The present study adopted Dugan’s (1991) definition of fecundity which states that fecundity refers to the

number of eggs present in a single clutch of an individual crab at the time of analysis (e.g., clutch size). The

ovigerous crabs were preserved in a mixture of ethanol, isopropyl alcohol, and acetone. Volumetric

determinations of fecundity were made on female crabs with newly extruded eggs (no eyespots evident upon

microscopic examination).

Litulo’s (2005) method of estimating fecundity was adapted in the study. Twenty (20) ovigerous females

with eggs were randomly selected for egg. Pleopods were removed from the females, placed in petri dishes

filled with water, and had their eggs detached by gradually adding a solution of sodium hypochlorite. Bare

pleopods were then discarded by being gently stirred in a beaker filled with 50 ml of seawater. With a pipette,

five sub-samples of 1 ml were taken from the water with eggs. The eggs in each sub-sample were counted

under a dissecting microscope. The average value obtained was extrapolated for the whole suspension in order

to estimate the number of eggs (Bezerraa and Matthews-Cascona, 2007).

An estimate of population fecundity was made for the whole sample. Population fecundity is defined in

this study as the number of eggs carried by a representative female crabs at or above the size of the smallest

ovigerous crab at the time of sampling. Pearson correlation was used to test the relationship between egg

volume and number of eggs with carapace length (CL, mm). To further analyze fecundity, data were analyzed

using the power function (Y = aX + b) of egg number (EN) vs. CL.

2.5 Statistical analysis

Data were analyzed using SPSS for Windows 11.0 (Copyright © SPSS Inc., 1989-2001). Descriptive statistics

(e.g., mean, mode, median, standard error of the mean, minimum, maximum, range, standard error of the sizes)

were calculated for the ovigerous, non-ovigerous and for male crabs for the purpose of comparison. T-test for

113

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

independent samples and one-way Analysis of Variance (ANOVA) were used to test significant differences

among groups (males vs. ovigerous females vs. non-ovigerous females; ovigerous vs. ovigerous) in terms of

mean carapace length (CL, mm). No estimates of density or abundance were made. To test the null hypothesis

that each group of sample comes from a normal distribution, one-sample Kolmogorov-Smirnov (KS)

procedure was used. This goodness-of-fit test assesses whether the observed cumulative distribution function

for a variable with a specified theoretical distribution, which in this case, normal distribution. Two-sample KS

test was also used to test the null hypothesis that male and female samples, as well as ovigerous and ovigerous

samples have the same distribution. Lastly, Pearson correlation was used to test the relationship between egg

volume and number of eggs with carapace length (CL, mm).

3 Results

3.1 Female size and age

There were more male Grapsus tenuicrustatus (52.94%, n = 81) than females (47.06%, n = 72) collected with

a sex ratio of 1:1.13. Thirty percent (30%, n = 32) of the samples were ovigerous females (Table 1).

Table 2 and Fig. 1 present the comparison of the carapace length (CL, mm) of the males and non-ovigerous

and ovigerous female G. tenuicrustatus. Ovigerous female crabs have the largest CL (mean ± SE: 31.25 ± 1.43)

compared with the males (mean ± SE: 22.14 ± 0.726) and non-ovigerous females (mean ± SE: 26.63 ± 1.12).

The differences were significant (F = 20.383, df = 2, p < 0.01). Difference between non-ovigerous and

ovigerous females was also found significant (t = 2.582, df = 70, p < 0.05). The CL (mm) of all samples

showed a mean ± SE of 25.22 ± 0.636. A sample with the smallest CL (12 mm) was found among male crabs

while the largest was from the ovigerous females (43 mm).

Fig. 2 (a-d) shows the size frequency distributions of all the crabs, males crabs only, female crabs only and

ovigerous females only, respectively. There was a non-conspicuous bi-modal size distribution for all the crabs,

with non-normal distributions for all crabs (KS = 1.735, p < 0.05) and for males (KS = 1.464, p < 0.05), but

not when all females or ovigerous females only were grouped together. When the size-frequency distribution

of males was compared with females, the distributions are significantly different from each other (KS = 2.582,

p < 0.0001). The same was observed between the size-frequency of ovigerous and non-ovigerous female crabs

(KS = 1502, p < 0.05).

Based on the size at maturity, and the minimum (5th percentile) and maximum (95th percentile) sizes of

ovigerous female crabs determined from size-frequency distributions for the whole sample, the largest among

the samples is an ovigerous female crab (CL = 43 mm) while the smallest is also from the same group

(CL=16). From the cumulative number of ovigerous crabs, the minimum and maximum sizes of ovigerous

female crabs are estimated as CL = 16.65 mm and CL= 42.35 mm, respectively (Table 3).

There are more ovigerous crabs belonging to the first year age class (CL = 16 - 33) (53.13 %) than those

that belong to the older class (CL = 34 – 43) (46.88 %). About 97% of the female ovigerous crabs are above

the size of the smallest crabs (CL = 17 – 43). Moreover, overlapping size modes were shown among the

ovigerous female crabs (23 mm, 34 mm, 35 mm (Table 4 and 5).

3.2 Population fecundity

The fecundity of G. tenuicrusttus ranged from 4400 (CL = 16 mm) to 26,400 (CL = 43 mm) eggs. Egg volume

ranged from 0.40 ml to 2.40 ml. The egg diameter ranged from 1.1 µm to 5.0 µm with an average diameter of

3.170 µm (Table 6; Fig. 4). There are more crabs belonging to CL class of 11-20 mm and 21-30 mm that carry

eggs at different stages (Table 7).

As shown in Table 8, egg count ranged from 4400 to 26400 with a mean of 12684 eggs. Forty-four percent

(44%) of the ovigerous crabs have eggs ranging from 4400 to 8800. Egg number was positively correlated with

114

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

female size (r = 0.794, n = 32) and the resulting scatter plot shows a linear trend (Fig. 3).

Table 1 Total number and sex ratios of collected G. tenuicrustatus at Talim Bay, Lian, Batangas.

Males Non-ovigerous females

Ovigerous females

Male and Female

Sex ratio

Total 81 40 32 153 1:1.13 Percentage 52.94 26.14 20.92 100

Table 2 Carapace length (CL, mm) of collected G. tenuicrustatus at Talim Bay, Lian, Batangas. Parameters

Males

Non- ovigerous females

Ovigerous females

All

n Mean ± SEa, b

S.D. Median Mode Minimum Maximum Range

81 22.14 ± 0.726 6.532 20 18 12 40 28

40 26.63 ± 1.12 7.080 24 23 14 42 28

32 31.25 ± 1.43 8.104 33 23c

16 43 27

153 25.22 ± 0.636 7.872 23 18 12 43 31

a. Difference between groups is significant based on ANOVA (F = 20.383, df = 2, p < 0.01). b. Difference between non-ovigerous and ovigerous females is significant based on t-Test for independent samples (t = 2.582, df = 70, p < 0.05). c. Multiple modes, the smallest value is shown.

Table 3 Size data for G. tenuicrustatus collected at Talim Bay, Lian, Batangas. Category of crab

Carapace length (CL, mm)

Largest male 40 Largest non-ovigerous female 42 Smallest ovigerous female 16 Largest ovigerous female 43 Minimum (5th percentile) sizes 16.65 Maximum (95th percentile) sizes 42.35

Table 4 Size at maturity of ovigerous female G. tenuicrustatus at Talim Bay, Lian, Batangas. Carapace Length

(CL, mm) Total %

Ovigerous crabs above the size of the smallest ovigerous crabs

17 – 43

31 96.88

Ovigerous crabs assigned to older age class

34 – 43

15 46.87

Ovigerous crabs assigned to the first year class

16 – 33

17 53.13

115

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

Table 5 Age classes of female G. tenuicrustatus collected at Talim Bay, Lian, Batangas based on modal breaks of Carapace Length (CL, mm) (n=32).

Age class Carapace Length (CL, mm)

Mode Total %

First year 16 – 33 23 17 53.13 Year 2 and 3 34 – 43 34, 35 15 46.87

Table 6 Egg volume (ml), number of eggs, and diameter of eggs sampled from collected G. tenuicrustatus at Talim Bay, Lian, Batangas.

Parameters

Carapace Length (CL, mm)

Egg volume (ml)

No. of Eggs

Egg Diameter (µm)

n Mean ± SE S.D. Median Mode Minimum Maximum Range

32 31.25±1.43a,b

8.104 33 23b

16 43 27

32 1.15±0.10a

0.592 1.05 0.60 0.40 2.40 2.00

32 12684.38±1153.04b

6522.572 11550.00 6600 4400 26400 22000

40 3.170±0.18 1.146 3.55 3.5 1.1 5.0 3.9

a, b. Positive correlation based on 2-tailed Pearson Correlation (r2 = 0.794, n =32).

Table 7 Numbers of ovigerous females of G. tenuicrustatus in different size groups (expressed as a percentage of total numbers of females collected) and relative number of females carrying various egg stages. Carapace width class (mm)

No. of females

Ovigerous females (%)

No. of ovigerous females carrying egg stage 1 2 3 4

11 - 20 5 60.0 5 4 4 2 21 - 30 7 71.4 6 5 5 0 31 - 40 8 87.5 5 0 1 1 41 - 50 3 100.0 0 1 0 0

Table 8 Estimate number of age in different size groups of ovigerous G. tenuicrustatus collected at Talim Bay, Lian, Batangas (n=32).

Estimate No. of Eggs Number of ovigerous females

%

4,400 – 8,800 14 43.75 8,801 – 13,200 6 18.75 13,201 – 17,600 4 12.5 17,601 – 22,000 4 12.5 22,001 – 26,400 4 12.5 Total 32 100

116

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

Fig. 1 Sizes of (a) male and female G. tenuicrustatus and (b) female ovigerous and non-ovigerous G. tenuicrustatus collected at Talim Bay, Lian, Batangas.

a

b

117

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

a

b

c

118

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

Fig. 2 Size-Frequency distribution of (a) all G. tenuicrustatus (n=153), (b) all the male G. tenuicrustatus (n=81), (c) all the female G. tenuicrustatus (n=72), and (d) all the ovigerous female G. tenuicrustatus (n=32) collected at Talim Bay, Lian, Batangas.

Fig. 3 Scatter plot for the relationship between egg number (EN) and female size (CL, mm) of G. tenuicrustatus collected at Talim Bay, Lian, Batangas.

d

119

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

Fig. 4 Eggs of G. tenuicrustatus collected at Talim Bay, Lian, Batangas. (a) egg still undivided, fully filled with yolk; (b to e), the free region of yolk is just visible; free area of yolk is conspicuously larger than earlier periods and ocular lobes are already visible; (f & g) overview of egg microscope.

a b

c d

g

e

f

120

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

4 Discussion

There are many reasons for the observed differences in total number collected and sex ratio among the G.

tenuicrustatus in the study site. First, in crustacean populations, sexual differences in distribution and mortality

may be responsible for unbalanced sex ratios (Johnson, 2003). Based on previous reports, differences between

male and female are not only exhibited by their spatial distributions and mortality rates but also on the effect of

predation on crab sex ratio (Montague, 1980; Wolf et al., 1975; Spivak et al., 1991).

Second, it was observed that the physiologic and behaviorally homeostatic crab populations living in

constant environments present a 1:1 sex ratio, or slightly male-biased. On the other hand, populations that

inhabit variable environments present deviations toward the females, in order to maximize the evolutionary

potential due to unequal selection between males and females (Geisel, 1972). Hence, it can be inferred that the

study site provided a rather male-biased environment wherein a more stable and constant conditions are

present.

In 1930, Fisher predicted that in random mating populations the evolutionary stable sex ratio would be 1:1.

Several studies supported this hypothesis. For instance, Bezerra and Matthews-Cascon (2007) found out that

the overall sex ratio of Uca thayeri population did not differ significantly from the expected 1:1 ratio and

therefore showed that this population is physiologic and behaviorally adapted to the habitat, besides also being

evolutionary stable.

Lastly, Costa (2000) observed that the low number of ovigerous females might also be due to the fact that

ovigerous females hide inside deep burrows in order to incubate their eggs. Thus, as observed in the present

study wherein the sex ratio did not differ significantly from the expected 1:1 ratio, indeed in a majority of

species is close to unity, despite some variations between populations of a species, and from year to year in the

same population (Nikolsky, 1963; Ofori-Danson, 1990).

Mantelatto and Fransozo (1996) explained that size at the onset of sexual maturity is a crucial variable to

be taken into account while investigating the reproductive ecology of a given organism. For crustaceans there

are not always outer characteristics such as color and size that clearly indicates when an individual reaches

sexual maturity In brachyuran crabs, this is easier in females due to unambiguous signals of breeding

competency, such as the presence of eggs attached to pleopods (Flores and Paula, 2002).

Among ocypodid crabs, sexual dimorphism is evidenced by males reaching larger sizes than females

(Lopez Greco et al., 2000). Females may have reduced somatic growth compared to males because they

concentrate their energy budget for gonad development. Moreover, males may reach larger sizes for successful

competition for copulation with more than one female, since larger male ocypodid crabs may have greater

chances of obtaining females for copulation and win more intra-specific fights (Christy and Salmon, 1984;

Christy, 1987). This may not be applicable in the case of G. tenuicrustatus, which was shown to have larger

females than males. Aside from reproductive pressure, other environmental and physiological factors might

explain why females are larger than males among G. tenuicrustatus.

Estimates based on the smallest egg-bearing female are dependent on the sample size and do not indicate

the average size at which females in a given population reach maturity (Lopez Greco and Rodriguez, 2004;

Ituarte et al., 2004). Hence, comparative studies using a morphological (macroscopic and histological) and

morphological analysis could be used for more precise estimates in determining the size at which males and

females reach maturity (Litulo, 2005).

The results also support the notion that size at sexual maturity and fecundity are the key parameters that

should reflect the lifetime investment in reproduction (Ramirez Llodra, 2002; Lopez Greco and Rodriguez,

2004). Certainly, fecundity and the size at the onset of sexual maturity of a species influence the periodicity

and duration of breeding season. Other important factors include temperature, salinity, food availability,

121

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

rainfall, photoperiod and lunar cycles. (Colpo and Negreiros-Fransozo, 2003; Costa and Negreiros-Fransozo,

2003; Litulo, 2004), but such variables were not explored in the present study and thereby merit further

investigation.

Fecundity is the number of eggs per female and a determinant of the reproductive potential of a species and

the stock size of its population (Mantelatto and Fransozo, 1997). It is an important parameter measured in

crustaceans for the estimation of the reproductive potential and future stock size of a given species or

population (Hattori and Pinheiro, 2001). Further, fecundity is directly related to life-history traits such as egg

size, age at maturity, life span and reproductive effort (Ramirez Llodra, 2002). The variation in fecundity is

very common in crab and has been reported by many workers like Erdman and Blake (1988); Melville Smith

(1987); Pauley et al. (1986); Hill et al. (1989); Gray (1969), and Ong (1966).

Hines (1982) explained that fecundity of crabs varies from species to species and also varies within the

same species due to different factors such as age, size, nourishment, ecological conditions of the water body

etc. Variation in fecundity was primarily a reflection of variation in the size of the crab at maturity.

The relationship between fecundity and size at sexual maturity depends on the life-history strategies of a

species (Ramirez Llodra, 2002). Earlier maturing species usually have a shorter generation because of the

shorter generation time needed to reach first reproduction and, the cost may be observed in a reduction in

future fecundity. In contrast, species with delayed maturity live longer, allowing them to grow larger and

therefore have a higher fecundity (Ramirez Llodra, 2002). Moreover, the longer lifespan may also give the

possibility of undergoing a higher number of lifetime fecundity and spawnings, which are characteristic in

tropical species (Emmerson, 1994).

In genus of Brachyurans, the Uca species, Thurman (1985) reported that the greatest egg amount (25012)

was registered for a female with 26.5mm of CW but concluded that the fecundity of the species in temperate

and tropical areas vary greatly, where the size and the amount of eggs are in close association with the

environmental conditions. More specifically in other studies, it was recorded that for a subtropical Uca thayeri

population that females from 23 to 26 of CW carried more than 45000 eggs, showing that in U. thayeri

fecundity is correlated with environmental conditions (Costa, 2000).

With the range of 3.9 mm in the diameter of eggs of G. tenuicrustatus as revealed by the findings of the

current study (see Table 6), the common observation that brachyuran show a great diversity of embryonic

development, especially owing to a significant variation in egg size (Hines, 1982) is supported. For blue crabs,

C. sapidus, a mean fecundity of 3.2 million eggs was revealed (Guillory et al., 1996). Shields et al. (1990)

mentioned that such variations in fecundity among brachyuran crabs may be caused by many factors including

climatic regimes, habitat and biological constraints.

Strong size-fecundity relationships are found in brachyuran families (Hines, 1982; Hartnoll, 1985). The

results of the present study are not an exception as shown by the results.

The results of the present study suggest that of an increase in number of eggs as the crabs grow larger. A

positive allometry between egg number and female size implies of an increase in fecundity of an increase of

female size. The relationship between female size and fecundity is a major characteristic of reproduction in

many crustaceans, and is related to morphological and physiological constraints in energy allocation and gonad

maturation (Ramirez Llodra, 2002). Litulo (2005) reported similar results for other brachyurans summarized

by Hines (1982) and studies done by Erdman and Blake (1988) on female golden crab, Geryon fenneri; Kumar

et al. (2000) on blue swimming crab, P. pelagicus, and Kyomo on sesarmid crab, Sesarma intermedia.

Carapace shape affects the volume reserved for gonadal development and spawn size (Hines, 1982;

Mantelatto and Fransozo, 1997; Koga, 1982). The allometric relationships between fecundity and crab size

variables is explained by the fact that egg mass is limited by the space available for the accumulation of

122

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

reserves as well as the gonadal development inside the cephalothorax of the crabs (Litulo, 2004). Similarly, in

the present study, it has been found that the number of eggs increased linearly with the increase of carapace

length.

5 Conclusion

The significant reproductive characteristics of G. tenuicrustatus in Talim Bay, Batangas include: (a) a slightly

male-biased ratio of 1:1.13; ovigerous female crabs having the largest CL compared with the males and non-

ovigerous females; more ovigerous crabs belonging to the first year age class than the older classes; fecundity

ranged from 4400 (CL = 16 mm) to 26,400 (CL = 43 mm) eggs; and the number of eggs increased with

increase in female size.

Acknowledgment

This work was supported by the scholarship awarded to the main author by the Advanced Science and

Technology Human Resource Development Program (ASTHRDP) of the Department of Science and

Technology (DOST) of the Philippines and Faculty and Staff Development Program of Ateneo de Naga

University, Philippines.

References

Bezerra LE, Matthews-Cascon H. 2007. Copulation and reproductive biology of the fiddler crab Uca thayeri

Rathbun, 1900 (Crustacea: Ocypodidae) in a tropical mangrove from Northeast Brazil. Acta Oecologica 31:

251-258

Christy JH, Salmon M. 1984. Ecology and evolution of mating system of fiddler crabs (genus Uca). Biological

Reviews, 59: 483-509

Christy JH. 1987. Female choice and breeding behavior of the fiddler crab Uca beebei. Journal of Crustacean

Biology, 7: 624-635

Colpo KD, Negreiros-Fransozo ML. 2003. Reproductive output of Uca vocator (Herbst, 1804) (Brachyura,

Ocypodidae) from three subtropical mangroves in Brazil. Crustaceana, 76: 1-11

Costa TM. 2000. Ecologia de caranguejos semiterrestres do geˆ nero Uca (Crustacea, Decapoda, Ocypodidae)

de uma a ́ rea de manguezal, em Ubatuba (SP). Ph.D. thesis, Universidade Estadual Paulista, Brazil

Costa TM, Negreiros-Fransozo ML. 2002. Population biology of Uca thayeri Rathbun, 1900 (Brachyura,

Ocypodidae) in a subtropical South America mangrove area: results from transect and catch-per-unit-

effort techniques. Crustaceana, 75: 1201-1218

Diesel R. 1989. Structure and function of the reproductive system of the symbiotic spider crab Inachus

phalangium (Decapoda: Majidae): Observations on sperm transfer, sperm storage and spawning. Journal of

Crustacean Biology, 9: 266-277

Dugan JE. 1990. Geographic and temporal variation in the lifehistory, growth and reproductive biology of the

sand crab, Emerita analoga (Stimpson). Ph.D. dissertation. University of California, Santa Barbara, USA

Dugan JE, Wenner A, Hubbard D. 1991. Geographic variation in the reproductive biology of the sand crab

Emerita analoga (Stimpson) on the California coast. Journal of Experimental Marine Biology and Ecology,

150: 63-81

Eickstaedt LL. 1969. The reproductive biology of the sand crab Emerita analoga (Stimpson). Ph.D.

Dissertation. Stanford University, Stanford, USA

Emmerson WD. 1994. Seasonal breeding cycles and sex ratios of eight species of crabs from Mgazana, a

mangrove estuary in Transkei, southern Africa. Journal of Crustacean Biology, 14: 568-578

123

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

Erdman RB, Blake NJ. 1988. Reproductive biology of female golden crabs Geryon fenneri Manning and

Holthuis, from souththeastern Florida. Journal of Crustacean Biology, 8: 392-400

Flores AVV, Paula J. 2002. Sexual maturity, larval release and reproductive output of two brachyuran crabs

from a rocky intertidal area in central Portugal. Invertebrate Reproduction and Development, 41: 21-34

Geisel JT. 1972. Sex ratio, rate of evolution, and environmental heterogeneity. American Naturalist, 106: 380-

387

George S. 1999. Egg quality, larval growth and phenotypic plasticity in a forcipulate seastar. Journal of

Experimental Marine Biology and Ecology, 237: 203-224

Gimé́nez L, Anger K. 2001. Relationships among salinity, egg size, embryonic development, and larval

biomass in the estuarine crab Chasmagnathus granulata Dana, 1851. Journal of Experimental Marine

Biology and Ecology, 260: 241-257

Gray GW Jr. 1969. Investigation of the Basic Life History of the Red Crab (Geryon quinquedens). Project 3-

46R Completion Report, Rhode Island Division of Conservation, USA

Guillory V, Prejean E, Bourgeois M, et al. 1996. A biological and fisheries profile of the blue crab, Callinectes

sapidus. LA. Department of Wildlife and Fisheries Management Plan Series, 8(1): 210

Hartnoll RG. 1985. Growth, sexual maturity and reproductive output. In: Factors in Adult Growth, Crustacean

Issues (Wenner AM, ed). 101-128, Balkema, Rotterdam, Netherlands

Hattori GY, Pinheiro MAA. 2001. Fecundity and embryology of Pachycheles milinifer (Dana, 1852)

(Anomura, Porcellanidae) at Praia Grande, Ubatuba, SP, Brazil. Nauplius, 9: 97-109

Hill J, Fowler DL, Van Den Avyle MJ. 1989. Species Profiles: Life Histories and Environmental

Requirements of the Coastal Fishes and Invertebrates (Mid-Atlantic)--Blue Crab. U.S. Fish and Wildlife

Service Biology Report 82 (11.100). U.S. Army Corps of Engineers, TR EL-82-4, USA

Hines AH. 1982. Allometric constraints and variables of reproductive effort in brachyuran crabs. Marine

Biology, 69: 309-320

Holland D. 1978. Lipid reserves and energy metabolism in the larvae of benthic marine invertebrates. In:

Biochemical and Biophysical Perspectives in Marine Biology (Vol. 4) (Malins D, Sargent J, eds). 85-123,

Academic Press, New York, USA

http://species-identification.org/species.php?species_group=crabs_of_japan&id=1656

Ituarte RB, Spivak ED, Luppi TA. 2004. Female reproductive cycle of the Southwestern Atlantic estuarine

crab Chasmagnathus granulatus (Brachyura: Grapsoidea: Varunidae). Scientia Marina, 68: 127-137

Jaeckle W. 1995. Variation in the size, energy content, and biochemical composition of invertebrate eggs:

correlates to the mode of larval development. In: Ecology of Marine Invertebrate Larvae (McEdward L,

ed). 49-77, CRC Press, Boca Raton, USA

Johnson PTJ. 2003. Biased sex ratios in fiddler crabs (Brachyura, Ocypodidae): a review and evaluation of the

influence of sampling method, size class and sex-specific mortality. Crustaceana, 76: 559-580

Koga T. 1998. Reproductive success and two modes of mating in the sand-bubbler crab Scopimera globosa.

Journal of Experimental Marine Biology and Ecology, 229: 197-207

Kumar M, Ferguson G, Xiao Y, et al. 2000. Studies on reproductive biology and distribution of blue swimmer

crab (Portunus pelagicus) in South Australian Waters. SARDI Research Report Series, 47

Levin L, Bridges T. 1995. Pattern and diversity in reproduction and development. In: Ecology of Marine

Invertebrate Larvae (McEdward L, ed). CRC Press, Boca Raton, USA

Litulo C. 2004. Fecundity of the Pantropical Fiddler Crab Uca annulipes (H. Milne Edwards, 1837)

(Brachyura: Ocypodidae) at Costa do Sol Mangrove, Maputo Bay, Southern Mozambique. WIOMSA, 3(1)

Litulo, C. 2005. Population structure and reproductive biology of the fiddler crab Uca inversa (Hoffman, 1874)

124

Arthropods, 2013, 2(3): 111-125

IAEES www.iaees.org

(Brachyura: Ocypodidae). Acta Oecologia, 27: 135-141

Lopez Greco LS, Hernandez JE, Bolanos J, et al. 2000. Population features of Microphrys bicornutus Latreille,

1825 (Brachyura, Majidae) from Isla Margarita,Venezuela. Hydrobiologia, 439: 151-159

Mantelatto F LM, Fransozo A. 1997. Fecundity of the crab Callinectes ornatus Ordway, 1863 (Decapoda,

Brachyura, Portunidae) from the Ubatuba region, São Paulo, Brazil. Crustaceana, 70: 214-224

Mantelatto FLM, FransozoA. 1996. Size at sexual maturity in Callinectes ornatus (Brachyura, Portunidae)

from the Ubatuba region (SP), Brazil. Nauplius, 4: 29-38

Melville-Smith R. 1987. The reproductive biology of Geryon maritae (Decapoda, Brachyura) off South West

Africa/Namibia. Crustaceana, 53: 259-275

Montague CL. 1980. A natural history of temperate western Atlantic fiddler crabs (Genus Uca) with reference

to their impact on the salt march. Contributions in Marine Science, 23: 25-55

Mouton ECJ, Felder DL. 1995. Reproduction of the fiddler crabs Uca longissignalis and Uca spinicarpa in a

Gulf of Mexico salt march. Estuaries, 18: 469-481

Nikolsky GV. 1963. The Ecology of Fishes. Academic Press, New York, USA

Ofori- Danson, P.K. 1990. Reproductive ecology of the trigger fish, Ballistes capriscus from the Ghanianain

coastal waters. Tropical Ecology, 31 (1): 1–11.

Ong KS. 1966. Observations on the post-larval life history of Scylla serrata Forskall, reared in the laboratory.

The Malaysian Agriculture Journal, 45: 429-443

Oshiro LMY. 1999. Aspectos reprodutivos do caranguejo guaia Menippe nodifrons Stimpson (Crustacea,

Decapoda, Xanthidae) da baia de Sepetiba, Rio de Janeiro, Brazil. Revista Brasileira de Zoologia, 16: 827-

834

Paschke K. 1998. Untersuchungen zum Energiestoffwechsel wa ̈hrend der Embryonalentwicklung der

Nordsee-Garnele Crangon crangon (Linnaeus 1758). Decapoda: Caridea.. PhD thesis. University of

Hamburg, Germany

Pauley GB, Amstrong DA, Heun TW. 1986. Species Profiles: Life histories and Environmental Requirements

of the Coastal Fishes and Invertebrates (Pacific-Northwest) Dungeness Crab. U.S. Fish and Wildlife

Service Biology Report 82 (11.63). U.S. Army Corps of Engineers, TR EL-82-4, USA

Ramirez Llodra E. 2002. Fecundity and life-history strategies in marine invertebrates. Advances in Marine

Biology, 43: 87-170

Sastry A. 1983. Ecological aspects of reproduction. In: The Biology of Crustacea (Vol. 8) (Bliss D, ed). 179-

269, Academic Press, New York, USA

Shields JD. 1992. The reproductive, ecology and fecundity of Cancer crabs. In: Crustacean Egg Production

(Wenner AM, Kurts AM, eds). Crustacean Issues 7. Balkema, Rotterdam, Netherlands

Spivak E, Gavio MA, Navarro CE. 1991. Life history and structure of the world’s southernmost Uca

population: Uca uruguayensis (Crustacea, Brachyura) in Mar Chiquita lagoon. Bulletin of Marine Science,

48: 679-688

Thurman, C.L., 1985. Evaporative water loss, corporal temperature and the distribution of sympatric fiddler

crabs (Uca) from south Texas. Comparative. Biochemistry and Physiology A, 119: 279-286

Wenner AM, Hubbard DM, Dugan JE, et al. 1987. Egg production by sand crabs (Emerita analoga) as a

function of size and year class (Decapoda, Hippidae). Biological Bulletin, 172: 225-235

Wenner AM, Fusaro C, Oaten A. 1974. Size at onset of sexual maturity and growth rate in crustacean

populations. Canadian Journal of Zoology, 52: 1095-1106

Wolf PL, Shanholtzer SF, Reimold RJ. 1975. Population estimates for Uca pugnax (Smith, 1870) on the

Duplin estuary marsh, Georgia, U.S.A. (Decapoda, Brachyura, Ocypodidae). Crustaceana, 29: 79-91

125

Arthropods, 2013, 2(3): 126-136

IAEES www.iaees.org

Article

Biochemical properties of digestive carbohydrases from the sugar

beet weevil, Lixus incanescens (Coleoptera: Curculionidae)

Seyed Mohammad Ahsaei, Vahid Hosseininaveh, Mahdieh Bigham Department of Plant Protection, College of Agriculture, University of Tehran, Karaj 31587-77871, Iran

E-mail: [email protected],[email protected]

Received 22 April 2013; Accepted 25 May 2013; Published online 1 September 2013

Abstract

The sugar beet weevil, Lixus incanescens B., is one of the most important pests of sugar beet plant in Iran. The

petioles and leaves of sugar beet are attacked by larvae and adults of the sugar beet weevil. Chemical

application is currently used for controlling the pest. Digestion in the alimentary canal of the sugar beet weevil

is facilitated by some carbohydrases. Results of the in vitro studies indicated the presence of α-amylase, β-

glucosidase and β-galactosidase in the digestive tract of the pest. Highest activities of α-amylase, β-glucosidase

and β-galactosidase were at pH 5, pH 5 and pH 4, respectively. No significant α-glucosidase and α-

galactosidase activity was detected in the pest’s digestive system. Optimum temperatures for α-amylase, β-

glucosidase and β-galactosidase activity were determined at 45, 50 and 40 °C, respectively. α-amylase was

more stable under acidic condition (pH 4 to pH 6) than under highly acidic and alkaline condition. Na+ and K+

increased α-amylase activity, but sodium dodecyl sulfate significantly decreased amylase activity. Also, the

activity of α-amylase was inhibited by the other compounds such as MgCl2, CaCl2 and EDTA. Zymogram

analysis using native-PAGE revealed one band of α-amylase activity in Lixus incanescens. High activity of

carbohydrases in the digestive system of adults was determined and further researches are needed to be applied

to design new strategies for controlling the sugar beet weevil based on natural carbohydrase inhibitors.

Keywords α-amylases; sugar beet weevil; carbohydrases; Lixus incanescens.

1 Introduction

The sugar beet weevil, Lixus incanescens B. (Coleoptera: Curculionidae) is a key pest of sugar beet plants

causing significant damage to living plants in Iran. It has been reported from many parts of Iran and some

other countries (Davatchi et al., 1960; Aleeva, 1953). The sugar beet weevil has three generations per year.

The petioles and leaves of sugar beet plants are attacked by larvae and adults of L. incanescens. The larvae can

cause up to 75% root weight loss (Ocete et al., 1994). The most damage happens in the second and third

generations. Loss in early-planted sugar beet is less than late-planted ones (Parvizi et al., 1988). Currently,


Arthropods, 2013, 2(3): 126-136

IAEES www.iaees.org

application of chemical pesticides has been a fundamental tool for pest control. However, it has had serious

consequences such as intoxication of animals and people, contamination of air, water, and soil, high

persistence in the environment, resistance in pests, and has impacted beneficial insects (Rodriguez et al., 1960;

Huignard et al., 2005). The use of such pesticides in urban areas holds special risks, as most pesticides are not

very selective, which has led to the search for safe and environmentally friendly alternatives (Breuer et al.,

2000). It seems that an integrated pest management (IPM) program, including application of selective

pesticides and some resistant cultivars bearing carbohydrase inhibitors as well as the use of field sanitation and

crop rotation would provide the best management option for control of this pest (Meiners et al., 1978).

Understanding the biochemistry of the enzymes present in the gut of target pests is the first step to design

inhibitor-transgenic crops (Oppert, 2000; Oppert et al., 2000; Wilhite et al., 2000). α-amylases (α-1,4-glucan-

4-glucanohydrolases; EC3.2.1.1) are the hydrolytic enzymes that there are in microorganisms, plants and

animals such as insects. α-amylases are hydrolytic enzymes that catalyze the hydrolysis of α-D-(1, 4) glucan

linkages in starch and related carbohydrates (Strobl et al., 1998; Ferreira et al., 1989). Α-amylases have been

detected in Coleoptera, Hymenoptera, Heteroptera, Orthoptera, Lepidoptera and Diptera (Hori, 1970;

Kanekatso, 1978; Baker, 1987; Baker, 1991; Terra et al., 1988; Schumaker et al., 1993; Ferreira et al., 1999).

Most insects depend on their amylases and glucosidases for efficient digestion of their diet. For both the

maintenance of adult longevity and optimal larval growth, carbohydrates are essential for initial hydrolyzing of

energy-producing nutrients (Dadd, 1985). α-glucosidases hydrolysis non-reducing 1,4-linked alpha-D-glucose

residues and releases α-D-glucose. α-glucosidase hydrolyzes several substrates, including maltose, sucrose ,

maltodextrin and pNP-α-Dglucopyranoside (Terra et al., 1996). β-glucosidases catalyzes hydrolysis of β-1,4

linkages between two glucoses or glucose-substituted molecules (Terra et al., 1996). α-galactosidases are exo-

acting glycoside hydrolases that cleave α-linked galactose residues from carbohydrates such as melibiose,

raffinose and gluco- or galactomannans (Meier et al., 1982). β-galactosidases are the enzyme that catalyses the

hydrolysis of β-galactosides into monosaccharides (Sezginturk et al., 2008). Our knowledge of β-

galactosidase in insects is still rudimentary.

The destructive effects of the pest L. incanescens on the production and handling of sugar beet plants have

made this insect an important target for biochemical study. There are significant variations among the

properties of insect digestive enzymes. To develop the control strategy utilizing plant-carbohydraceous

inhibitors, it is necessary to have more information on the gut enzymatic activities of insects (Wilhite et al.,

2000). In the current study, we have carried out a detailed biochemical analysis of digestive carbohydrolytic

activity in L. incanescens adults.


2.1 Test insect

Adults of the sugar beet weevil, Lixus incanescens, were collected from the fields of sugar beet plants at

Qazvin province, Iran and were used as the enzyme source for subsequent experiments.

2.2 Sample preparation

Whole gut of the adults were removed by dissection under a stereo-microscope in ice-cold saline buffer, and

homogenized in distilled water using a hand-held glass homogenizer. The homogenates were centrifuged at

16,000 × g for 15 min at 4°C. Resulted supernatants were removed and kept at -20 °C until needed.

2.3 α-amylase assay

α-amylase activity was assayed using dinitrosalicylic acid (DNSA) as the reagent (Strobl et al., 1998) and 1%

soluble starch (Merck) as the substrate. Ten microliters of the enzyme source was added to 5 μl substrate and

85 μl of the universal buffer (0.02 M; sodium citrate- phosphate-borate). The reaction mixture was incubated at

127

Arthropods, 2013, 2(3): 126-136

IAEES www.iaees.org

35°C for 30 min. Thereafter, 100 μl DNSA was added to the reaction mixture and heated in boiling water for

10 min. After cooling the mixture, the absorbance was measured at 540 nm. All assays were performed in

triplicate.

2.4 Glucosidase and galactosidase assay

Glucosidase and galactosidase assays was carried out by incubating 10 μl of the enzyme extract with 85 μl of

50 mM universal buffer (pH 4 to pH 11; sodium citrate-phosphate-borate) and 5 μl of each pnitrophenyl- α-D-

glucopyranoside (pNPαGlu, 5 mM) for α-glucosidase or p-nitrophenyl-β-D-glucopyranoside (pNPβGlu, 5 mM)

for β-glucosidase at 35 °C for 30 min. After the addition of NaOH (2 M) to the reaction mixture, released p-

nitrophenol was determined by measuring the absorbance at 405 nm. In the blanks, enzyme extract was added

to the reaction mixture after the addition of NaOH. The same procedure was used for detecting α- and β-

galactosidase activity but with p-nitrophenyl-α-D-galactosidase (pNPαGal, 5 mM) and p-nitrophenyl-β-D-

galactosidase (pNPβGal, 5 mM), respectively, as the substrates.

2.5 Effect of pH and temperature on enzyme activity

The effects of temperature and pH on the activity of α-amylase, β-glucosidases and β-galactosidase were

examined using enzymes extracted from the whole gut. Effect of temperature on α-amylase, β-glucosidases

and β-galactosidases activity was determined by incubating the reaction mixture at 20, 30, 35, 40, 45, 50, 60

and 70 °C for 30 min followed by measurement of the enzyme activity. The optimum pH for α-amylase, β-

glucosidases and β-galactosidase activity was determined using the universal buffer (sodium citrate-phosphate-

borate) at a pH range 3 to pH 11 with one degree interval.

2.6 pH stability of α-amylase

Stability of α-amylase was determined over a broad pH range and two incubation time periods. Enzyme extract

was mixed with buffer and incubated for 1 and 10 hr at 37°C. The substrate was then added to the buffered

enzyme extract, and α-amylase activity was determined as described above.

2.7 Effect of activators and inhibitors on α-amylase

To investigate the effect of several salts on α-amylase activity, assays were performed in the presence of

different concentrations of K+, Na+, Ca2+, and Mg2+ chloride salts as well as sodium dodecyl sulfate (SDS) and

Ethylenediaminetetraacetic acid (EDTA). The enzyme sample was pre-incubated with the compounds for 15

min.

2.8 Zymogram analysis

Activity of α-amylase present in the crude homogenates of the whole gut of the adults was visualized using

native polyacrylamide gel electrophoresis (native-PAGE). Native-PAGE was performed in a 10% (w/v)

resolving gel and a 4% stacking gel. The sample buffer contained 25% stacking buffer (0.5 M Tris–HCl; pH

6.8), 20% glycerol, 0.005% (w/v) bromophenol blue, but no mercaptoethanol and no boiling. After

electrophoresis, the gel was rinsed in distilled water and incubated for 1 h in phosphate buffer containing 1%

starch and 20 mM CaCl2 at pH 5. The gel was then rinsed with distilled water and incubated with a solution of

10 mM I2 and 14 mM KI to stop the reaction and to stain the unreacted starch background. Areas bearing α-

amylase activity appeared as light bands against a dark background.

2.9 Protein determination

Protein content was determined using the method of Lowry et al. (1951). Bovine serum albumin was used as

the standard.

3 Results

3.1 α-amylase activity

The effect of pH on amylolytic activity from the whole gut extracts is shown in Fig. 1. Relatively higher

128

Arthropods, 2013, 2(3): 126-136

IAEES www.iaees.org

activities were detected over a broad range of acidic condition (pH 4 to pH 6) with maximum activity at pH 5.

Fig. 1 The effect of pH on the activity of α-amylase extracted from the digestive system of L. incanescens adults.

Fig. 2 The effect of pH on the activity of β-glucosidases extracted from the digestive system of L. incanescens adults.

Fig. 3 The effect of pH on the activity of β-galactosidases extracted from the digestive system of L. incanescens adults.

129

Arthropods, 2013, 2(3): 126-136

IAEES www.iaees.org

3.2 Glucosidase activity

β-glucosidase activity in the gut increased steadily from pH 3 to pH 6, and then decreased with increased pH

above 6 (Fig. 2). The optimal pH for β-glucosidase activity occurred at 5.0. Activity for α–glucosidase was not

significantly detected in digestive system of L. incanescens adults.

3.3 Galactosidase activity

β-Galactosidase activity increased steadily from pH 3 to pH 4, then decreased with increased pH above 4. The

optimal pH for β-galactosidase activity occurred at pH 4.0 (Fig. 3). Activity for α–galactosidase was not

detected in digestive system of Lixus incanescens adults.

3.4 Effect of temperature on α-amylase activity

The optimum temperature for α-amylase activity was 45 °C (Fig. 4). Digestive β-glucosidase was optimally

active at 50 °C (Fig. 5). Optimum temperature for β-galactosidase activity was determined at 40 °C (Fig. 6).

Fig. 4 The effect of temperature on the activity of α-amylase extracted from the digestive system of L. incanescens adults.

Fig. 5 The effect of temperature on the activity of β-glucosidases extracted from the digestive system of L. incanescens adults.

130

Arthropods, 2013, 2(3): 126-136

IAEES www.iaees.org

Fig. 6 The effect of temperature on the activity of β-galactosidases extracted from the digestive system of L. incanescens adults.

Fig. 7 Stability of digestive α-amylase from L. incanescens at different pHs after 1 and 10 hrs incubation period.

3.5 pH stability of α-amylase

The results revealed that α-amylase was more stable in acidic to slightly acidic condition (pH 4 to pH 6) with a

short incubation period (1 hr; Fig. 7). However, α-amylase stability decreased under alkaline condition and a

longer time incubation period (10 hr; Fig. 7).

3.6 Effect of activators and inhibitors on α-amylase activity

Na+ and K+ enhanced α-amylase activity, whereas sodium dodecyl sulfate (SDS) significantly decreased α-

amylase activity. Activity of α-amylase was inhibited by other compounds such as MgCl2, CaCl2 and

ethylenediaminetetraacetic acid (EDTA) (Fig. 8).

3.7 Zymogram analysis

Further characterizations of the α-amylase activity of the gut extract from the adults of L. incanescens using

starch as the substrate under native-PAGE are shown in Fig. 9. At least one band of α-amylase activity was

revealed from the gut extract.

131

Arthropods, 2013, 2(3): 126-136

IAEES www.iaees.org

Fig. 8 Effects of different compounds on the midgut digestive amylase activity in L. incanescens adults.

Fig. 9 Zymogram of α- amylase extracted from the digestive system of L. incanescens.

132

Arthropods, 2013, 2(3): 126-136

IAEES www.iaees.org

4 Discussion

Carbohydrases play a vital role in food digestion in insects. The present study clearly showed that α-amylase,

β-glucosidase and β-galactosidase are active in the gut of L. incanescens adults. The enzymes are optimally

active in acidic condition which is congruent with the pH prevailing in the gut of Coleoptera. Stability of

amylase was also consistent with the pH value of the midgut and showed its maximum stability at acidic to

slightly acidic condition in L. incanescens. The optimal pHs for hemolymph and gut α-amylases of the red

palm weevil, Rhynchophorus ferrugineus Olivier (Col.: Curculionidae) were 5 to 6 and 4 to 5, respectively

(Saberi et al., 2012). The optimum pH for digestive α- amylase activity in the salivary glands and midgut of

Brachynema germari Kol. was determined at 6 and 5, respectively (Ramzi et al., 2010). In insects, amylases

are usually most active in neutral to slightly acidic condition (Baker, 1983; Terra et al, 1996). The optimal pH

values in larvae of various coleopterans were from 4 to 5.8 for amylases (Baker, 1983). The optimum activity

for α-amylase from the midgut of Hypera postica larvae was determined at pH 5.0 (Vatanparast et al., 2010).

Hypera postica α-amylase was more stable at pH 5 to pH 6 than highly alkaline and acidic pH in larval midgut

of the pest (Vatanparast et al., 2010). The pH in the tissues from which the enzymes are isolated usually

corresponds to the optimum pH for digestive enzyme activity. Biochemical properties of many partially

purified or purified insect gut α- glucosidases are known, and regardless of the corresponding midgut pH value,

their optimal pH range from 5 to 6.5 (Terra et al., 1994). Activity for α–glucosidase and α–galactosidase were

not detected in digestive system of Lixus incanescens adults. Insect β-glucosidases have pH optima of 4.5 to

6.5 (Terra et al, 1994; Azevedo et al, 2003). β-glucosidase activity has been reported in Diatraea saccharalis

Fabricius (Lepidoptera: Pyralidae) (Azevedo et al., 2003), Parnassius apollo ssp. frankenbergeri (Lepidoptera:

Papilionidae) (Nakonieczny et al, 2006). Also, digestive β-glucosidase activity has been reported in

Rhynchophorus palmarum L. (Coleoptera: Curculionidae) (Yapi et al., 2009). In Dysdercus peruvianus Guerin

(Hemiptera: Pyrrochoridae), galactosidases have an optimum pH of 5.0 (Silva et al., 1997). In Abracris

flavolineata De Geer (Orthoptera: Acrididae) midguts, there are two α-galactosidases and these enzyme have

maximum activity at pH 5.4 (Ferreira et al., 2001). β-galactosidase activity has been detected in Spodoptera

frugiperda (Marana et al., 2000) and Diatraea saccharalis, Tenebrio molitor (Franco et al., 2000). The highest

activities of α- and β-glucosidases of Rhynchophorus ferrugineus were at pH 5 and of α- and β-galactosidases

at pH 4 (Saberi et al., 2012). Data on the biochemical characterization of β-galactosidase in insects are not

complete.

Maximum carbohyrolytic activity was obtained at 40 to 50 °C congruent with the other insects. Higher gut

α-amylase activity of the red palm weevil was obtained at 40 to 50 °C (Saberi et al., 2012). Digestive α-

amylase from H. postica showed its maximal activity at 35 °C (Vatanparast et al., 2010). For α-amylase

activity, optimum temperature was 60 °C in Bombyx mori L. (Lepidoptera: Bombycidae) (Kanekatso, 1978).

In midgut of Brachynema germari Kol. (Hemiptera: Pentatomidae) optimum temperature for α- and β-

galactosidase was 30 °C (Ramzi et al., 2010). The sugar beet weevil β-glucosidase has optimum temperature

activity at 50 °C (Fig. 5). Also, the optimal temperature for β-galactosidase in the digestive system this pest

was 40 °C (Fig. 6). Alpha and β-glucosidases and α- and β-galactosidases from the red palm weevil had an

optimum temperature activity at 50, 50–60, 40–60, and 40 °C, respectively (Saberi et al., 2012). Activity of

digestive α- and β-glucosidases٫ α- and β-galactosidases in the digestive system of Xanthogaleruca luteola

were optimum at 60, 50, 40, 60 °C , respectively (Sharifi et al., 2011).

In the presence of Ca2+, Mg2+ and SDS, digestive α-amylase activity of Hypera postica significantly

decreased, while Na+ and K+ did not significantly affect the enzyme activity (Vatanparast et al., 2010).

Zymogram analysis showed the presence of one band of α-amylase activity in the gut of L. incanescens similar

to other coleopteran pests (Vatanparast et al., 2010). However, zymogram analysis revealed that red palm

133

Arthropods, 2013, 2(3): 126-136

IAEES www.iaees.org

weevil hemolymph and gut α-amylases had two isoforms (Saberi et al., 2012).

Producing pest-resistant transgenic plants help us to decrease the use of chemical pesticides and the

entering of these chemicals in the environment. Understand digestive enzymes of the target insect is first step

in designing a controlling program based on inhibition of digestion (Strobl et al., 1998). The biochemical

characterization of insect digestive enzymes will greatly help to design new strategies for insect control and

will facilitate the understanding of the mechanisms responsible for the inhibitory potential of the

carbohydraceous plant inhibitors.

Acknowledgements

The authors appreciate Seyed Mohammad Tabadkani and Alireza Badieneshin for their great helps with this

work. The authors have no conflict of interests to declare.

References

Aleeva MN.1953. Data on the biology of weevils (Col.: Curculionidae) injurious to sugar beet in Kazakhstan.

Ent Obozon, 33: 103-108 (In Russian)

Azevedo TM, Terra WR, Ferreira C.2003. Purification and characterization of three β-glycosidases from

midgut of the sugar cane borer, Diathraea saccharalis. Insect Biochemistry and Molecular Biology, 33:

81-92

Baker JE.1983. Properties of amylase from midgets of larvae of Sitophilus zeamais and Sitophilus granaries.

Insect Biochemistry, 13: 421-428

Baker JE.1987. Purification of isoamylases from the rice weevil, Sitophilus oryzae (L.) (Coleoptera:

Curculionidae) by high-performance liquid chromatography and their interaction with partiallypurified

amylase inhibitors from wheat. Insect Biochemistry, 17: 37-44

Baker JE.1991. Purification and partial characterization of α-amylase allozymes from the lesser grain borer,

Rhyzopertha dominica. Insect Biochemistry, 21: 303-311

Breuer M, De loof A. 2000. Laboratory trials with NeemAzal-T/S on the allergenic forest pest Thaumetopoea

processionea L. In: Practice Oriented Results on Use and Production of Neem-ingredients and Pheromones

VIII (Kleeberg H, Zebitz CPW, eds). 23-29, Hohensolms, Germany

Dadd RH. 1985. Nutrition: organisms. In: Comprehensive Insect Physiology Biochemistry and Pharmacology

8 (Kerkut GA, Gilbert LI, eds). 338-341, Pergamon Press, Oxford, UK

Davatchi A, Kheyri M. 1960. The Most Important Pest of Sugar Beet and Their Control. The Plan

Organization, Tehran, Iran

Ferreira C, Terra WR. 1989. Spatial organization of digestion, secretory mechanisms and digestive enzyme

properties in Pheropsophus aequinoctialis (Coleoptera: Carabidae). Insect Biochemistry, 19: 383-391

Ferreira C, Marana SR, Silva C, et al. 1999. Properties of digestive glycosidases and peptidases and the

permeability of the peritrophic membranes of Abracris flavolineata (Orthoptera: Acrididae). Comparative

Biochemistry and Physiology B, 123: 241-250

Ferreira AHP, Marana SR, Terra WR, et al. 2001. Purification, molecular cloning, and properties of a β-

glycosidase isolated from midgut lumen of Tenebrio molitor (Coleoptera) larvae. Insect Biochemistry and

Molecular Biology, 31: 1065-1076

Franco OL, Riggen DJ, Melo FR, et al. 2000. Activity of wheat α-amylase inhibitors towards bruchid α-

amylases and structural explanation of observed specificities. European Journal of Biochemistry, 267:

2166-2173

134

Arthropods, 2013, 2(3): 126-136

IAEES www.iaees.org

Hori K. 1970. Some properties of amylase in the salivary gland of Lygus disponsi (Hemiptera). Journal of

Insect Physiology, 16: 373-386

Huignard J, Dugravot S, Ketoh KG, et al. 2005. Use of secondary plant products to protect the seeds of a

legume, cowpea: Effects on insect pests and their parasitoids. In: Biopesticides of Plant Origin (Regnault-

Roger C, Philogène B, Vincent C eds). 123-137, Lavoisier, Paris, France

Kanekatso R. 1978. Studies on further properties for an alkaline amylase in the digestive juice of the silkworm,

Bombyx mori. Journal of Fac. Text. Science and Technology, 76: 1-21

Lowry OH, Rosembrough NJ, Farr AL, Randdall RJ.1951. Protein measurement with the Folin phenol reagent.

Journal of Biological Chemistry, 193: 267-275

Marana SR, Terra WR, Ferreira C. 2000. Purification and properties of a β-glycosidase purified from midgut

cells of Spodoptera frugiperda (Lepidoptera) larvae. Insect Biochemistry and Molecular Biology, 30:

1139-1146

Meier H, Reid JSG. 1982. Reserve polysaccharides other than starch in higher plants. In: Encyclopedia of

Plant Physiology (Loewus FA, Tanner W, eds). 418-471, Springer, New York, USA

Meiners JP, Elden TC. 1978. Resistance to insects and diseases in Phaseolus. In: Advances in Legume Science.

International Legume Conference (Summerfield RS, Bunting AH, eds). 359-364, Kew Surrey, UK

Nakonieczny M, Michalczyk K, Kedziorski A. 2006. Midgut glycosidases activities in monophagous larvae of

Apollo butterfly, Parnassius apollo ssp. frankenbergeri. C. R. Biology, 329: 765-774

Ocete R, Ocete ME, Perez-izquierdo MA , et al. 1994. Approximation to the phenology of Lixus junci B. (Col.:

Curculionidae) in La Rioja Alta: estimate of the damage it causes. Boletín de Sanidad Vegetal Plagas, 20:

611-616

Oppert B. 2000. Transgenic plants expressing enzyme inhibitors and the prospects for biopesticide

development. In: Advances in Biopesticide Research (Koul O, Dhaliwal GS, eds). 83-95, Hardwood

Academic, Amsterdam, Netherlands

Oppert B, Hartzer K, Smith CM.2000. Digestive proteinases of alfalfa weevil, Hypera postica (Gyllenhal)

(Coleoptera; Curculionidae). Transactions of Kansas Academy of Science, 103(3-4): 99-110

Parvizi R, Javanmoghaddam H. 1988. Investigation on some biological features of the sugar beet weevil (Lixus

incanescens B.). Ent Phyt Appliq., 55(1-2): 1-8 (In Persian)

Ramzi S, Hosseininaveh V. 2010. Biochemical characterization of digestive α-amylase, α-glucosidase and β-

glucosidase in pistachio green stink bug, Brachynema germari Kolenati (Hemiptera: Pentatomidae).

Journal of Asia-Pacific Entomology, 13: 215-219

Rodriguez C, Silva G, Vendramim YJ. 2003. Insecticidas de origen vegetal. In: Bases Para el Manejo Racional

de Insecticidas (Silva G, Hepp R, eds). 89-111, Universidad de Concepción, Fac. Agron. Chillán, Chile

Saberi riseh N, Ghadamyari M. 2012. Biochemical characterization of α-amylases from gut and hemolymph of

Rhynchophorus ferrugineus Olivieri (Col.: Curculionidae) and their inhibition by extracts from the

legumes Vigna radiata L. and Phaseolus vulgaris L. Research Report ISJ, 9: 72-81

Saberi riseh N, Ghadamyari M, Motamediniya B. 2012.Biochemical Characterisation of α- and β-glucosidases

and α- and β-galactosidases from Red Palm Weevil, Rhynchophorus ferrugineus (Olivier) (Col.:

Curculionidae). Plant Protection Science, 2: 85-93

Schumaker TTS, Cristofoletti PT, Terra WR. 1993. Properties and compartmentalization of digestive

carbohydrases and proteases in Scaptotrigona bipunctata (Apidae: Meliponinae) larvae. Apidologie, 24: 3-

17

Sezginturk MK, Dinckaya E. 2008. β-Galactosidase monitoring by a biosensor based on Clark electrode: Its

optimization, characterization and application. Biosensors and Bioelectronics, 23: 1799-1804

135

Arthropods, 2013, 2(3): 126-136

IAEES www.iaees.org

Sharifi M, Ghadamyari M, Mahdavi M, et al. 2011. Biochemical characterization of digestive carbohyrases

from Xanthogaleruca luteola and inhibition of its a-amylase by inhibitors extracted from the common bean.

Archives of Biological Science Belgrade, 63(3): 705-716

Silva CP, Terra WR. 1997. Alpha-galactosidase activity in ingested seeds and in the midgut of Dysdercus

peruvianus (Hemiptera: Pyrrhocoridae). Archives of Insect Biochemistry and Physiology, 34: 443-460

Strobl S, Maskos K, Wiegand G, et al. 1998. A novel strategy for inhibition of α-amylases: yellow meal worm

α-amylase in complex with Ragi bifunctional inhibitor at 2.5 Å resolution. Structure, 6: 911-921

Terra WR, Espinoza-fuentes EP, Ferreira C. 1988. Midgut amylase, lysozyme, aminopeptidase, and trehalase

from larvae and adults of Musca domestica. Archives of Insect Biochemistry and Physiology, 9: 283-297

Terra WR, Ferreira C. 1994. Insect digestive enzymes: properties, compartmentalization and function.

Comparative Biochemistry and Physiology B, 109: 1-62

Terra WR, Cristofoletti T. 1996. Midgut proteinases in three divergent species of Coleoptera. Comparative

Biochemistry and Physiology B, 113: 725-730

Terra WR, Ferreira C, Jordan BP, et al. 1996. Digestive enzymes. In: Biology of the Insect Midgut (Lehane MJ,

Billingsley PF, eds). 153-194, Chapman & Hall, London, UK

Vatanparast M, Hosseininaveh V. 2010. Digestive amylase and pectinase activity in the larvae of alfalfa weevil

Hypera postica (Coleoptera: Curculionidae). Entomological Research, 40: 328-335

Wilhite SE, Elden TC, Brzin J, et al. 2000. Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil

midgut with biochemical and plant-derived proteinase inhibitors. Insect Biochemistry and Molecular

Biology, 30: 1181-1188

Yapi DYA, Gnakri D, Niamke SL, et al. 2009. Purification and biochemical characterization of a specific β-

glucosidase from the digestive fluid of larvae of the palm weevil, Rhynchophorus palmarum. Journal of

Insect Science, 9: 4

136

Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

Article

Induced plant resistance as a pest management tactic on piercing

sucking insects of sesame crop

M. F. Mahmoud Plant Protection Department, Faculty of Agriculture, Suez Canal University, 41522 Ismailia, Egypt


Received 5 April 2013; Accepted 10 May 2013; Published online 1 September 2013

Abstract

Sesame, Sesamum indicum L. is the most oil seed crop of the world and also a major oil seed crop of Egypt.

One of the major constraints in its production the damage caused by insect pests, particularly sucking insects

which suck the cell sap from leaves, flowers and capsules. Impact of three levels of potassin-F, salicylic acid

and combination between them on reduction infestation of Stink bug Nezara viridula L., Mirid bug

Creontiades sp., Green peach aphid Myzus persicae (Sulzer), Leafhopper Empoasca lybica de Berg and

Whitefly Bemisia tabaci (Gennadius) of sesame crop cultivar Shandawil 3 was carried out during 2010-2011

crop season at Experimental farm, Faculty of Agriculture, Suez Canal University, Ismailia, Egypt. Also, the

impacts of potassin-F and salicylic acid on yield production of sesame were studied. Results indicated that

percent of reduction of infestation by N. viridula, M. persicae, Creontiades sp., E. lybicae, B. tabaci and

phyllody disease were significantly higher at Level 2 (Potassin-F= 2.5 cm/l, Salicylic acid= 10−3 M and

Potassin + Salicylic= 2.5 cm/l + 10−3 M) and consequently higher seed yield per plant were obtained.

Keywords Sesamum indicum; potassin-F; salicylic acid; induced resistance; piercing sucking insects.

1 Introduction

Sesame (Sesamum indicum L.) is one of the important oil crops. It is cultivated in almost all tropical and sub

tropical Asia and African countries for its highly nutritious and edible seeds (Iwo et al., 2002). In Egypt,

sesame is considering a food crop rather than oilseed crops because most of its seeds consumed directly. It is

clear that the increase in sesame production during last ten years was mainly due to the increase in its growing

area, especially in newly reclaimed sandy soils.

Among various factors responsible for the low productivity levels of sesame, the insect pests associated

with flowering phase usually inflicts very severe damage to the crop. The physical damage may be less than

those of foliage pests, yet their impact on final yield is colossal.

Piercing sucking insects have great economic importance to sesame plants. They cause serious damage


Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

directly by sucking plant sap or indirectly by transmission of virus and mycoplasma diseases (El-Gindy, 2002).

Stink bug Nezara viridula L., Mirid bug Creontiades sp., Green peach aphid Myzus persicae (Sulzer),

Leafhopper Empoasca lybica de Berg and Whitefly Bemisia tabaci (Gennadius) are serious pests which suck

the cell sap from leaves, flowers and capsules. Due to this curling of leaf margins downward, reddening of leaf

margins, stunted growth of the plants, sickly appearance of the crop and subnormal growth of the leaf tissue

occur. The peculiar yellow spots are found on upper surface of leaves affected by whitefly. Jassid and whitefly

are also responsible to transmit phyllody and leaf curl diseases in sesame, respectively. Induced plant

resistance is very important component of integrated pest management programs, including adequate inorganic

fertilization kinds and amounts. Of all the nutrients that affect plant diseases and pests, potassium (K) is

probably the most effective. It has been considered a key component of plant nutrition that significantly

influences crop growth and some pests infestation. There are limited information about results of potassium

fertilization on reduction population of piercing sucking insects of sesame. Potassium fertilizer is negatively

associated with occurrence of Aphis glycines (Myers and Gratton, 2006) and leafhoppers and mites (Parihar

and Upadhyay, 2001). Cotton aphid population density at seedling stage was suppressed by potassium

fertilizers in proper rate (Ai et al., 2011). The high rates of potassium reduced the population density of some

piercing sucking insects on cereal, legumes and maize plants (El-Gindy, 2002, 2006).

Inducible defenses play a major role in conferring disease resistance against plant pathogens (Maleck and

Dietrich, 1999) and their effects on phytophagous insects can include increased toxicity, delay of larval

development or increased attack by insect parasitoids (Baldwin and Preston, 1999). Inducible defenses are

thought to compromise plant fitness less and may be more durable than constitutive defense mechanisms

(Agrawal, 1998). The plant nutrient status in an indicator of host plant quality plays an important role in the

population dynamics of many herbivores (Sarwar et al., 2011). Salicylic acid has been shown to have a

positive effect on some species of plants with regards to expression of dormant genes. However, no work has

yet been done to assess the level of insect attacks on the sesame plants treated with salicylic acid. Considering

the fact that some substances in association with crops under particular conditions can induce the presence of

both major and minor pests and diseases at different stages of development, the level of sucking insect

infestations on sesame in association with salicylic acid under our prevalent conditions must be properly

studied and understood. The concept of integrated pest management has taken centre stage in pest and diseases

management on a wide range of crops. This approach includes an integration of cultural, biological, chemical

and host plant resistance methods in controlling pests. Therefore, the purpose of this research was to

investigate the impact of three levels of potassin-F fertilizer and salicylic acid on reduction densities and

infestation of piercing sucking insects attacking sesame plants within two successive seasons, and to

investigate the impact of these treatments on yield production of sesame crop. Finding out the effect of

treatments on the pest load and damage can help update the control strategy of sucking insects on sesame.


2.1 Experimental design

This experiment was carried out at the Experimental farm, Faculty of Agriculture, Suez Canal University,

Ismailia, Egypt during the growing seasons of sesame (2010 and 2011). The soil texture of experimental site

was sandy soil (94.5% sand, 2.5% silt and 3.0% clay) with pH of 7.8.

Seeds of cultivar of sesame namely (Shandawil 3) were purchased from the Agriculture Research Center,

Giza, Egypt. These seeds were treated with Rizolex-T (3 g/kg seeds) before planting to prevent rot infection. A

randomized complete block experimental design was used and each experimental unit measuring an area of 3.5

× 4 m2. Three treatments were used at three levels and control.

138

Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

Table 1 Potassin-F and salicylic treatment levels used in the present study.

Levels Treatments Potassin –F (P) (0 N: 8P: 30K)

Salicylic Acid (S)

*P + S

Control Level 1 Level 2 Level 3

0.0 1cm/l 2.5 cm/l 4 cm/l

0.0 10 −2 M 10 −3 M 10 −4 M

0.0 1cm/l + 10 −2 M 2.5 cm/l + 10 −3 M 4 cm/l +10 −4 M

* P= Potassin- F foliar application, S= Salicylic foliar application.

2.2 Sampling of studied insects

2.2.1 Jassid and aphid

25 sesame leaves were sampled weekly from each plot early in the morning from three different levels of the

plant. The upper and lower surfaces of the randomly chosen sesame leaves were carefully examined using lens

(5×) to count all individuals of Empoasca lybica and Myzus persicae and the data were recorded (El-Zahi et

al., 2012).

2.2.2 Whitefly

25 sesame leaves were randomly chosen from each plot and picked up weekly from the levels mentioned

above. The chosen leaves were transmitted to the laboratory in paper bags where binocular-microscope was

used to count the immature stages of B. tabaci (nymphs and pupae). The duration of sampling as mentioned

before.

2.2.3 Stink and mirid bugs

Sampling methods were made directly in the field on five randomly selected plants from each plot. All stages

were recorded. The reduction percentage of insect infestations was calculated for the seasons of 2010 and 2011.

2.3 Treatments

Sesame plants were sprayed with three levels of potassin–F and salicylic acid separately and together.

Potassin-F (P) was purchased from the Agriculture Research Center, Giza and Salicylic acid (S) was initially

dissolved in a few drops of dimethylsulfoxide and the final volume was reached using water. The

concentrations of (S) with a surfactant triton 0.1% and concentrations of (P) were sprayed twice on the whole

foliage of sesame (after 30 days from sowing and at the initiation of flowering stage). One set served as control

was sprayed with water only. The plants were sprayed with hand spray bottles10 liter capacity.

2.4 Effect of potassin-F fertilizer (P) and salicylic acid (S) on sesame production

Some parameters were measured as follows: no. of capsule/ plant, capsule weight, no. of seed/capsule, weight

of 1000 seed and seed yield/plant of sesame (Dhurve, 2008).

1- Number of capsule per plant: the number of capsule on plant was counted and expressed as capsule per

plant. Observation was made on 10 randomly selected plants per treatment.

2- Capsule weight: weight of 25 randomly selected capsules from each treatment was measured before the

harvest using an electronic balance.

3- Number of seeds per capsule: the number of seeds in a capsule was counted and expressed as seeds per

capsule. Observation was made on 10 randomly selected plants per treatment.

4- Weight of 1000 seeds: the observation was made by weighing 1000 dried seeds drawn randomly from

each treatment using an electronic balance.

5- Seed yield per plant: after maturation, the capsules from ten plants from each treatment were removed

and recorded. The seeds were separated and weight using an electronic balance and expressed in grams per

plant. Each treatment was replicated four times.

139

Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

2.5 Statistical analysis

Data obtained in all presented experiments were subjected to analysis of variance (ANOVA) and correlation

coefficient with the honestly significant difference value calculated as Tukey’s statistic at α = 0.05 (SAS

Institute, 2002).

3 Results

Results from Fig. 1 showed five piercing sucking insects attacked sesame during growing season 2010/2011

and phyllody and healthy plant of sesame. Data presented in Fig. 2 and 3 showed the effect of three levels of

potassin-F, salicylic acid and the combination between them on population density of some piercing sucking

insects and the mean number of phyllody disease for sesame cultvar shandawil 3, during the growing season of

2010 and 2011. Data in Fig. 2 and 3 revealed that the three levels of all treatments led to reduction of

population density of jassid (Empoasca lybica), aphid (Myzus persicae), stink bug (Nezara virdula), whitefly

(Bemesia tabaci), mirid bug (Creontiades sp.) and the mean number of phyllody disease. Also, data in Fig. 2

and 3 indicated that Level 2= (Potassin-F= 2.5 cm/l, Salicylic acid= 10−3 M and Potassin + Salicylic= 2.5 cm/l

+ 10−3 M) was the more affect on reduction of population density than level 1 and level 3. Moreover, the

combination of potassin-F and salicylic acid in level 2 was the most suitable treatment to reduce population of

insects and phyllody disease.

The relationship between the reduction of infestation, % and the three levels of treatments potassin-F,

salicylic acid and the combination between them for the growing seasons of sesame, 2010 and 2011 were

computed as a coefficient of determination based on the simple linear regression Fig. 4 and 5.

Data presented in Fig. 4 indicated that level 2 of treatments (P= 2.5 cm/l, S= 10−3 M and P+S= 2.5 cm/l +

10−3 M) gave high reduction of infestation for the following insects, B. tabaci, N. viridula, and M. persicae.

The coefficient of determination (R2) was 0.993, 0.931 and 0.1, respectively. Whenever, level 1 of treatments

(P= 1 cm/l, S= 10−2 M and P+S= 1 cm/l + 10−2 M) gave high reduction of infestation of E. lybica and

Creontiades sp. R2 was 0.923 and 0.750, respectively. Finally, the level 3 (P= 4 cm/l, S= 10−4 M and P+S= 4

cm/l + 10−4 M) gave high reduction of infestation only in phyllody disease at R2 = 0.999.

Also, data presented in Fig. 5 showed that level 2 of treatments (P= 2.5 cm/l, S= 10−3 M and P+S= 2.5

cm/l + 10−3 M) gave high influence on the reduction of infestation,% for the following insects, E. lybica, N.

viridula, Creontiades sp. and M. persicae. The coefficient of determination (R2) was 0.993, 0.931, 0.1 and

0.964, respectively. While, level 3 of treatments (P= 4 cm/l, S= 10−4 M and P+S= 4 cm/l + 10−4 M) gave high

reduction of infestation of B. tabaci and phyllody disease. Results further proved that R2 for B. tabaci and

phyllody disease in the infestation of reduction was very high at values of 0.964 and 0.750, respectively.

140

Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

Fig. 1 Some piercing sucking insects attacked sesame during growing season 2010/2011, 1- Empoasca lybica, 2- Bemesia tabaci, 3- Creontiades sp., 4- Myzus persicae, 5a- Nezara virdula eggs, 5b- N. virdula nymphs, 5c, N. viridula adults, 6- phyllody disease, 7- phyllody and healthy plant of sesame.

3

1 2

5 a 5 b 5 c

6 7

4

141

Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

05

10152025

303540

4550

C P S P+S C P S P+S C P S P+S

Level 1 Level 2 Level 3

Treatments

Mea

n n

o. o

f ja

ssid

/25

leav

es

0

20

40

60

80

100

120

140

160



Treatments

Mea

n n

o. o

f ap

hid

/ 25

leav

es

0

10

20

30

40

50

60



Treatments

Mea

n n

o. o

f st

ink

bu

g /

5 p

lan

ts

0

10

20

30

40

50

60

70



Treatments

Mea

n n

o. o

f w

hit

efly

/ 25

leav

es

02468

101214161820



Treatments

Mea

n n

o. o

f m

irid

bu

g /

5 p

lan

ts

02468

1012141618



Treatments

Mea

n n

o. o

f p

hyl

lod

y / 1

00p

lan

ts

Fig. 2 Effect of three levels of potassin-F, Salicylic acid and the combination between them on the mean number of some piercing sucking insects which attack sesame and the mean number of phyllody disease during the growing season of 2010. C= Control, P= Potassin-F, S= Salicylic and P+S= Potassin-F + Salicylic; Level 1= (P= 1 cm/l, S= 10−2 M and P+S= 1 cm/l + 10−2 M); Level 2= (P= 2.5 cm/l, S= 10−3 M and P+S= 2.5 cm/l + 10−3 M); Level 3= (P= 4 cm/l, S= 10−4 M and P+S= 4 cm/l + 10−4 M).

142

Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

0

10

20

30

40

50



Treatments

Mea

n n

o. o

f ja

ssid

/ 25

le

aves

0

20

40

60

80

100

120



Treatments

Mea

n no

. of

aphi

d / 2

5 le

aves

0

10

20

30

40

50



Treatments

Mea

n n

o. o

f st

ink

bu

g / 5

p

lan

ts

0

10

20

30

40

50

60



Treatments

Mea

n n

o. o

f w

hit

efly

/ 25

le

aves

02468

1012141618



Treatments

Mea

n n

o. o

f m

irid

bu

g / 5

p

lan

ts

0

5

10

15

20

25



Treatments

Mea

n n

o. o

f p

hyl

lod

y / 1

00

pla

nts

Fig. 3 Effect of three levels of potassin-F, Salicylic acid and the combination between them on the mean number of some piercing sucking insects which attack sesame and the mean number of phyllody disease during the growing season of 2011. C= Control, P= Potassin-F, S= Salicylic and P+S= Potassin-F + Salicylic; Level 1= (P= 1 cm/l, S= 10−2 M and P+S= 1 cm/l + 10−2 M); Level 2= (P= 2.5 cm/l, S= 10−3 M and P+S= 2.5 cm/l + 10−3 M); Level 3= (P= 4 cm/l, S= 10−4 M and P+S= 4 cm/l + 10−4 M).

143

Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

Fig. 4 Relationship between reduction of infestation, % and different levels of potassin-F, salicylic acid and the combination between them in sesame during growing season of 2010.

Creontiades sp.

Myzus persicae

Nezara viridula Bemesia tabaci

Empoasca lybica

Phyllody

144

Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

Fig. 5 Relationship between reduction of infestation, % and different levels of potassin-F, salicylic acid and the combination between them in sesame during growing season of 2011.

Creontiades sp.

Nezara viridula Bemisia tabaci

Empoasca lybica Myzus persicae

Phyllody

145

Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

Results in Table 2 showed the influence of potassin-F, salicylic acid and the combination between them on

number of capsule/ plant, capsule weight, number of seed/capsule, weight of 1000 seed and seed yield (g)/

plant of sesame. Data showed significant differences in some quantitative parameters either in 2010 or 2011.

Number of capsule/ plant, capsule weight (g) and seed yield (g)/ plant in all treatments were significantly

higher compared to control in both growing seasons. In 2010 season (df= 4,35; F= 1.315; P≤ 0.0009), (df=

4,35; F= 6.381; P≤ 0.0001), (df= 4,35; F= 2.793; P≤ 0.0166), respectively. While in 2011 season (df= 4,35; F=

6.440; P≤ 0.0000), (df= 4,35; F= 4.234; P≤ 0.0013), (df= 4,35; F= 8.432; P≤ 0.0000), respectively. Number of

seed/ capsule was insignificant compared to control in both seasons. It was (df= 4,35; F= 1.315; P≤ 0.2701),

(df= 4,35; F= 1.917; P≤ 0.0878), respectively. On the other hand, weight of 1000 seed was not significant in

2010 season (df= 4,35; F= 2.271; P≤ 0.0445), and it was significant in 2011 season (df= 4,35; F= 2.708; P≤

0.0194).

The correlation coefficient (r) between seed yield (g)/ plant which produced from sesame plants treated

with different levels of potassin, salicylic and the combination between them and reduction of infestation of

different piercing sucking insects and phyllody disease was calculated of both studied seasons and presented in

Table 3. Data in this Table showed greatest direct effect on seed yield (g)/ plant produced from sesame plants

treated with potassin, with reduction of infestation of M. persicae, E. lybica, B. tabaci and N. viridula in 2011

season. It was 0.961, 0.522, 0.956 and 0.918, respectively. Also, results showed direct effect on seed yield (g)/

plant produced from sesame plants treated with salicylic, with reduction of infestation M. persicae, E. lybica

and N. viridula in 2010 season. It was 0.971, 0.642 and 0.988, respectively. And it was 0.537 with Creontiades

sp. in 2011 season. The strength of a relationship between seed yield (g)/ plant and reduction of phyllody

disease was high in both growing seasons of sesame especially with combined treatment (potassin + salicylic).

It was (0.581) in 2010 and (0.841) in 2011, respectively.

On the other hand, most of negative correlation has been recorded between seed yield (g)/ plant produced

from sesame plants treated with combined levels of potassin and salicylic and reduction infestation of M.

persicae, E. lybica, N. viridula and Creontiades sp.

Table 2 Influence of potassin-F, salicylic acid and the combination between them on no. of capsule/ plant, capsule weight, no. of seed/capsule, weight of 1000 seed and seed yield/plant of sesame.

Treatments No. of capsule/ plant Capsule weight (g) No. of seed/capsule Weight of 1000 seed

Seed yield (g)/plant

2010 2011 2010 2011 2010 2011 2010 2011 2010 2011 Control (0) 68.50 c 67.25 c 1.83 c 1.85 c 62.50 a 63.00 a 4.45 a 4.50 b 19.05 b 19.06 d Potassin- F

L1** 71.25 bc 71.75 bc 1.91 bc 2.07 abc 64.25 a 60.25 a 4.46 a 4.47 b 21.81 ab 19.26 cd

L2 77.75 abc 84.00 ab 2.00 abc 2.15 ab 68.00 a 61.00 a 4.51 a 4.52 b 23.85 ab 23.12 bcd

L3 7 7.75 abc 78.75 abc 2.08 ab 2.14 ab 66.00 a 62.50 a 4.47 a 4.57 ab 23.28 ab 22.54 bcd

Salicylic

L1 78.75 abc 75.75 abc 1.98 abc 1.99 bc 63.75 a 64.50 a 4.51 a 4.60 ab 22.53 ab 22.59 bcd

L2 81.75 a 87.75 a 2.05 ab 2.27 a 69.00 a 68.75 a 4.52 a 4.77 a 25.53 a 28.82 a

L3 81.25 a 81.50 ab 2.12 a 2.08 abc 70.00 a 68.25 a 4.58 a 4.65 ab 25.98 a 25.86 a Potassin- F + Salicylic

L1 77.25 abc 79.00 abc 2.05 ab 1.96 bc 65.00 a 62.75 a 4.57 a 4.60 ab 22.99 ab 22.83 bcd

L2 81.75 a 86.50 a 2.16 a 2.15 ab 63.25 a 64.75 a 4.50 a 4.57 ab 23.26 ab 25.59 ab

L3 79.75 ab 83.00 ab 2.10 ab 2.02 abc 64.00 a 63.50 a 4.53 a 4.57 ab 23.23 ab 24.08 abc

LSD 0.5 - 6.056 7.381 0.114 0.166 6.507 6.131 0.083 0.147 3.319 2.931

*Means followed by the same letter in a column are not statistically different by Tukey's HSD (P=0.05) ** Level 1= (P= 1 cm/l, S= 10−2 M and P+S= 1 cm/l + 10−2 M); Level 2= (P= 2.5 cm/l, S= 10−3 M and P+S= 2.5 cm/l + 10−3 M); Level 3= (P= 4 cm/l, S= 10−4 M and P+S= 4 cm/l + 10−4 M).

146

Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

Table 3 Correlation coefficient (r) between seed yield (g)/plant produced from sesame plants treated with different levels of potassin-F, salicylic acid and the combination between them and reduction infestation of different piercing sucking insects and phyllody disease.

Seed yield (g)/plant

M. persicae E. lybica B. tabaci N. viridula

Creontiades sp.

Phyllody disease

2010 2011 2010 2011 2010 2011 2010 2011 2010 2011 2010 2011

Potassin-F 0.606 0.961 0.485 0.522 0.671 0.956 -0.949 0.918 -0.966 -0.986 -0.068 0.036

Salicylic 0.971 0.687 0.624 0.466 0.369 0.828 0.988 0.750 -0.039 0.537 0.150 -0.461

Potassin + Salicylic

-0.598 0.207 -0.547 0.281 0.561 0.281 -0.330 -0.047 -0.827 0.281 0.581 0.841

4 Discussion

In the present study, we applied potassin-F which consists of (0 Nitrogen: 8 Phosphorus: 30 Potassium). It is

well known that, potassium plays an important physiological roles including building up of resistance to insect

pests by influencing tissue of cell structures and biochemical processes. Potassium nutrition has a profound

effect on the profile and distribution of primary metabolites in plant tissues, which in turn could affect the

attractiveness of plant for insects and pathogens as well as their subsequent growth and development the plant

(Amtmann et al., 2008). A thicker cuticle in plants can be considered as a first line of defense to disease and

insect attack and increases the resistance to insect feeding, especially against sucking insects (Khattab, 2007).

Several studies conclusively indicate that phosphate is effective in controlling some important plant diseases

caused by pathogens. Action of phosphate anion is based on two mechanisms: the first is a direct toxic action

on the pathogen and the second in indirect action due to phosphite anion activates plant defence responses

(Cook et al., 2009; Avila et al., 2012).

Salicylic acid (SA), is a plant phenolic, is widely distributed throughout the plant kingdom. It is a

hormone-like substance, which plays an important role in the regulation many aspects of plant growth and

development (Raskin, 1992). However, it is especially famous for its ability to induce systemic acquired

resistance (SAR) in plants (Ryals et al., 1996) i.e. resistance in induced but also uninduced distal leaves of the

same plant.

Piercing sucking insects play an important role in reducing the production of sesame yield. The present

results revealed that sesame plants at the studied location was infested with Stink bug Nezara viridula L.,

Mirid bug Creontiades sp., Green peach aphid Myzus persicae (Sulzer), Leafhopper Empoasca lybica de Berg

and Whitefly Bemisia tabaci (Gennadius). These insects were recorded on sesame crop variety shandawil 3

during growing season of 2010 and 2011, Ismailia, Egypt (Mahmoud, 2012). Also, some of these pests were

recorded on potato plants by Parihar et al. (1996) and Kuroli (2001). On the other hand, M. percicae, A.

gossypii, A. craccivora, B. tabaci and Empoasca sp. populations were most at zero potassium fertilization in

comparison with 100, 150 and 200 kg/feddan, respectively (El-Gindy et al., 2009). Also, phyllody disease

plays a significant role in reducing yield of sesame. Leafhopper transmitted phytoplasma is the cause of

phyllody disease (Akhtar et al., 2008; Akhtar et al., 2009), which can cause up to 80% yield loss with disease

incidence of 61-80% (Kumar and Mishra, 1992).

Results in the present study confirmed significant effect of potassin-F fertilization on the insect infestations.

These findings are in agreement with those reported by (Kindler and Staples, 1970) who found that increasing

amount of potassium and phosphorus fertilizer, increased resistance against spotted alfalfa aphid in alfalfa,

green bug in sorghum (Schwessing and Wilde, 1979). Data in presented study indicated that Salicylic acid had

positive effects on plant growth, reduction infestation, % of M. persicae, B. tabaci, E. lybica, Creontiades sp.,

147

Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

N. viridula, phyllody disease and yield components and hence could find roles to play in integrated pest

management on sesame plants.

In general, treatment of potassin-F and salicylic acid separately and together, especially at the level 2

clearly caused significant decrease in population density and percentage of infestation of N. viridula,

Creontiades sp., M. persicae, E. lybica and B. tabaci. Also, it caused reduction in the mean number of

phyllody disease during 2010 and 2011 crop seasons of sesame. Moreover, it caused significant increase in the

number of capsules on sesame stem, capsule weight (g), thousand seed wt (g) and seed yield/ plant (g).

Therefore, using potassin-F fertilization and salicylic acid is so necessary for encouraging the defense system

of plant to be more resistance or tolerant to insect injury caused by piercing sucking insects. Elden and

Kenworkthy (1995) found that the optimum or higher levels of potassium nutrition have been implicated with

decline in the incidence of disease and insect pests in several plant species.

In conclusion, potassin-F and salicylic acid provided good nutrition and resistance for the reduction of pest

load and damage and consequently, enhanced seed yield of sesame cultivar (Shandawil 3) in the reclaimed

land. Application of potassin-F (P) and salicylic acid (S) separately or combined at level 2 (P= 2.5 cm/l, S=

10−3 M and P+S= 2.5 cm/l + 10−3 M) provided the best nutrition for effective reduction of sesame pest load

and damage with increased seed production. The findings of this study also recommended that even though

application of potassin-F and salicylic acid at the level 2 (P= 2.5 cm/l, S= 10−3 M and P+S= 2.5 cm/l + 10−3 M)

reduced pest load and increased the quantity and quality of sesame, higher seed yield could be enhanced

through integrated pest management, especially if this rate is applied in conjunction with other cultural

practices such as crop rotation, inter-cropping, planting date manipulation, plant spacing and biological control.

References

Agrawal AA. 1998. Induced responses to herbivory and increased plant performance. Science, 279: 1201-1202.

Ai TC, Liu ZY, Li CR, et al. 2011. Impact of fertilization on cotton aphid population in Bt. cotton production

system. Ecological Complexity, 8: 9-14

Akhtar KP, Dickinson M, Sarwar G, et al. 2008. First report on the association of a 16SrII phytoplasma with

sesame phyllody in Pakistan. Plant Patholology, 57: 771

Akhtar KP, Sarwari G, Dickinson M, et al. 2009. Sesame phyllody disease: its symptomatology, etiology, and

transmission in Pakistan. Turkish Journal of Agriculture, 33: 477-486

Amtmann A, Troufflard S, Armengaud P. 2008. The effect of potassium nutrition on pest and disease

resistance in plants. Plant Physiology, 133: 682-691

Ávila FW, Faquin V, Lobato AKS, et al. 2012. Growth, phosphorus status, and nutritional aspect in common

bean exposed to different soil phosphate levels and foliar-applied phosphorus forms. Scientific Research

and Essays, 7(25): 2195-2204

Baldwin IT, Preston CA. 1999. The eco-physiological complexity of plant responses to insect herbivores.

Planta, 208: 137-145

Cook PJ, Landschoot PJ, Schlossberg MJ. 2009. Inhibition of Pythium spp. and suppression of Pythium Blight

of turfgrasses with phosphonate fungicides. Plant Disease, 93(8): 809-814

Dhurve SS. 2008. Impact of honey bee population on seed production of niger. MSc thesis. University of

Agriculture Sciences, Dharwad, India

Edlin TC, Kenworthy WJ. 1995. Physiological responses of an insect-resistance soubean line to light and

nutrient stress. Journal of Economic Entomology, 88(2): 430-436

El-Gindy MA, El-Refaey RM, Abou Hatab EE. 2009. Abundance of some potato homopterous pests as

148

Arthropods, 2013, 2(3): 137-149

IAEES www.iaees.org

affected by potassium fertilization level. Egyptian Academic Journal of Biological Sciences, 2(2): 179-185

El-Gindy MA. 2002. Studies on certain homopterous insect vectors of plant pathogenic diseases. PhD thesis.

Faculty of Agriculture, Zagazig University, Zagazig, Egypt

El-Gindy MA. 2006. Susceptibility of three maize cultivars to leaf hopper infestations and effect of potassium

fertilizers on leaf hoppers. Egyptian Journal of Applied Sciences, 21(10A): 302-314

El-Zahi ES, Arif SA, Jehan BA, et al. 2012. Inorganic fertilization of cotton field-plants in relation to sucking

insects and yield production components of cotton plants. Journal of American Science, 8(2): 509-517

Iwo GA, Idowu AA, Ochigbo AA. 2002. Sesame genotypes for field stability and selection in Nigeria,

Nigerian Agricultural Journal, 33: 76-82

Khattab H. 2007. The Defense Mechanism of cabbage plant against phloem-sucking aphid (Brevicoryne

brassicae L.). Australian Journal of Basic and Applied Sciences, 1(1): 56-62

Kindler SA, Staples R. 1970. Nutrients and the reaction of two alfalfa clones to the spotted alfalfa aphid.

Journal of Economic Entomology, 63: 938-940

Kumar P, Mishra US. 1992. Diseases of Sesamum indicum in Rohilkhand: intensity and yield loss. Indian

Phytopathology, 45: 121-122

Kuroli G. 2001. Change of population density of the leafhoppers Empoasca solani Curtis and E. decipiens

Paoli feeding on potatoes. Növényvédelem, 37: 225-230

Mahmoud MF. 2012. Insects associated with sesame (Sesamum indicum L.) and the impact of insect

pollinators on crop production. Pesticides and Phytomedicine, 27(2): 117-129

Maleck K, Dietrich RA. 1999. Defense on multiple fronts: how do plants cope with diverse enemies? Trends in

Plant Science, 4: 215-219

Myers SW, Gratton C. 2006. Influence of potassium fertility on soybean aphid, Aphid glycines Matsumura

(Hemiptera: Aphididae), population dynamics at a field and regional scale. Environmental Entomology, 35:

219-227

Parihar SBS, Upadhyay NC. 2001. Effect of fertilizers (NPK) on incidence of leafhoppers and mite in potato

crop. Insect Environment, 7: 10-11

Parihar SBS, Verma KD, Malik K. 1996. Evaluation of potato genotypes against Aphis gossypii damage, Insect

Environment, 2: 48-49

Raskin K. 1992. Role of salicylic acid in plants. Annual Review of Plant Physiology and Plant Molecular

Biology, 43: 439

Ryals JA, Neuenschwander UH, Willits MG, et al. 1996. Systemic acquired resistance. Plant Cell, 8: 1809-

1819

Sarwar M, Ahmad N, Tofique M. 2011. Impact of soil potassium on population buildup of aphid (Homoptera:

Aphididae) and crop yield in canola (Brassica napus L.) field. Pakistan Journal of Zoology, 43(1): 15-19

SAS Institute Inc. 2002. SAS/STAT User’s Guide (Version 9.1). SAS Institute Inc, North Carolina, USA

Schwessing FC, Wilde G. 1979. Temperature and plant nutrient effects on resistance of seedling sorghum to

the green bug. Journal of Economic Entomology, 72: 20-23

149

Arthropods

Arthropods account for more than 65% of global species and 85% of animal species. On a temperate grassland,

arthropods hold a huge biomass (1,000kg/ha), seconded to plant (20,000kg/ha) and microorganisms (7,000kg/ha)

but much higher than mammals (1.2kg/ha), birds (0.3kg/ha), and nemantodes (120kg/ha). Arthropods play the

role of both pests and beneficial organisms. Some arthropods are important crop pests but others are natural

enemies. Some arthropods are important health pests but many crustaceans are important food sources of

humankinds. Arthropods govern the structures and functions of natural ecosystems, but are always ignored by

researchers. On the global scale, the surveys of mammals, birds and vascular plants were relatively perfect

because they were economically important and easily surveyed. However, arthropods, despite their ecological

and economical importance, have not yet been fully surveyed and recorded due to their difficulties to be

sampled. The research on arthropods must be further promoted. The journal, Arthropods, is inaugurated to

provide a public and appropriate platform for the publication of studies and reports on arthropods.

ARTHROPODS (ISSN 2224-4255) is an international journal devoted to the publication of articles on various

aspects of arthropods, e.g., ecology, biogeography, systematics, biodiversity (species diversity, genetic diversity,

et al.), conservation, control, etc. The journal provides a forum for examining the importance of arthropods in

biosphere (both terrestrial and marine ecosystems) and human life in such fields as agriculture, forestry, fishery,

environmental management and human health. The scope of Arthropods is wide and embraces all

arthropods-insects, arachnids, crustaceans, centipedes, millipedes, and other arthropods. Articles/short

communications on new taxa (species, genus, families, orders, etc.) and new records of arthropods are

particularly welcome.

Authors can submit their works to the email box of this journal, [email protected]. All manuscripts

submitted to Arthropods must be previously unpublished and may not be considered for publication elsewhere

at any time during review period of this journal.

In addition to free submissions from authors around the world, special issues are also accepted. The organizer of

a special issue can collect submissions (yielded from a research project, a research group, etc.) on a specific

topic, or submissions of a conference for publication of special issue.

Editorial Office: [email protected]

Publisher: International Academy of Ecology and Environmental Sciences

Address: Flat C, 23/F, Lucky Plaza, 315-321 Lockhart Road, Wanchai, Hong Kong

Tel: 00852-6555 7188

Fax: 00852-3177 9906


Arthropods ISSN 2224-4255 Volume 2, Number 3, 1 September 2013

Articles

The locomotory rhythmic activity in scorpions: with a review

Michael R. Warburg 95-104

Two records of Macrophthalmus Desmarest, 1823 (Decapoda: Brachyura: Thoracotremata)

from the NW of the Arabian Gulf

Amaal Gh. Yasser, Ibtisam M. AbdulSahib, Murtada D. Naser, et al. 105-110

Reproductive characteristics of a brachyuran crab, Grapsus tenuicrustatus (Herbst, 1783)

(Decapoda: Grapsidae) found in Talim Bay, Batangas, Philippines

Michael A. Clores, Gliceria B. Ramos 111-125

Biochemical properties of digestive carbohydrases from the sugar beet weevil, Lixus

incanescens (Coleoptera: Curculionidae)

Seyed Mohammad Ahsaei, Vahid Hosseininaveh, Mahdieh Bigham 126-136

Induced plant resistance as a pest management tactic on piercing sucking insects of sesame

crop

M. F. Mahmoud 137-149

The International Academy of Ecology and Environmental Sciences (IAEES) is a nonprofit and registered

international organization. It devotes to promote global ecology and environmental sciences and protect global

ecological environments, by publishing scientific publications, conducting research activities, launching

environmental programs, disseminating knowledge and technologies, sponsoring conferences, and providing

information and discussion spaces, etc. The responsibility for these publications rests with the International

Academy of Ecology and Environmental Sciences.

IAEES http://www.iaees.org/

Arthropods, 2013, Vol. 2, Iss. 3

Documents

Transcript of Arthropods, 2013, Vol. 2, Iss. 3