Observations on the life history of giant water bugs Lethocerus Mayr , 1853 ( Heteroptera : Nepomorpha : Belostomatidae ) in the Gangetic plains of India and Nepal

Acknowledgements: We thank the University Grants Commission (New Delhi, India), Central University of Bihar (Patna, India) , Dr. K. Venkataraman, Director, Zoological Survey of India, Kolkata, R.K. Sinha (Department of Zoology, P.U., Patna, India), Ram Kumar (Centre for Environmental Science, CUB, Patna, India), Deep Narayan Shah and Ram Devi Tachamo Shah (Department of Limnology and Nature Conservation, Senckenberg Research Institutes and Natural History Museums, Gelnhausen, Germany) for their kind help in fieldwork; Herbert Zettel (Entomological Department, Natural History Museum Vienna, Austria) for valuable support, encouragements and discussion.


INTrOduCTION
Aquatic bugs of the infraorder Nepomorpha are hemimetabolous insects.The juvenile stages (nymphs) are similar to the adult forms but they are distinguished by the successive growth of their wing pads.The nymphs are fully aquatic whereas many of the winged mature Nepomorpha can fly.This is an important strategy in the life cycle for active dispersal and colonization of new habitats.The largest arthropods in freshwaters are mostly represented by decapods Crustaceans like crabs, prawns and crayfish but several aquatic bugs are noticeably robust insects of an unusually large size with raptorial forelegs.Belostomatidae are named giant water bugs with about 160 accepted species worldwide of three subfamilies (Polhemus & Polhemus 2008;Morse 2009;Henry 2009).Some members of the subfamily Lethocerinae reach 109-115 mm total body length, the maximum size of adult aquatic insects (Perez-Goodwyn 2006).Their distribution range includes the circumtropical, subtropical and warm-temperate zones of all continents.The family Belostomatidae also shares several characters with the definition of "living fossils" since similar representations of these bugs are already known as fossil documentations from the Upper Triassic of Virginia (North America) since 210 Million years ago (Andersen & Weir 2004).The oldest Lethocerinae are found in the Jurassic of Germany with Mesobelostomum depertitum (Germar, 1839) (Ponomarenko 1985;Popov 1992).At present three extant genera Benacus Stål, 1861 (monotypic), Kirkaldyia Montandon, 1909 (monotypic), and Lethocerus Mayr, 1853 (22 species) are recognized with 24 species.The highest diversity with 17 species is observed in the neotropical region (Perez-Goodwyn 2006;Choate 2011).Records of tropical Asian Lethocerinae species are scattered in the literature (Easton & Wing-Wah Pun 1997;Sharma 1998;Bal & Basu 2002;Nahar 2004;Chen et al. 2005;Lekprayoon et al. 2007;Nesemann et al. 2012), only Thirumalai (2007) has summarized the distribution for India.Giant water bugs of the genus Lethocerus are traditionally used as human food in South-East Asia.
Lethocerinae appear to be a well-studied group of large-sized insects which is reflected in a comparatively high number of articles and book chapters dealing with their biology (Dudgeon 1999;Deckert & Göllner-Scheiding 2003;Polhemus & Polhemus 2008;Morse 2009;Henry 2009).Perez-Goodwyn (2006: 7) stated: "The reproductive behavior is peculiar in the family and different to that of Belostomatinae.The female lays her eggs outside the water ("emergent brooders" according to Smith 1997) on a vegetal substratum (sticks, so far as known, but L. mazzai chooses moss at the border of the ponds as substratum, see De Carlo, J.M. 1962).The male guards the eggs until hatching, keeping them moist and defending them from predators."Biological observations of four American and two Oriental species are published, namely Lethocerus americanus (Leidy, 1847) (Hoffman 1924;Hungerford 1925), Lethocerus maximus De Carlo, 1938(Cullen 1969), Lethocerus mazzai De Carlo, 1961(De Carlo 1969), Lethocerus medius (Guérin-Méneville, 1857) (Smith & Larsen 1993) and Lethocerus indicus (Lepeletier & Serville 1825) (Hoffman 1933a,b).The behavior of Kirkaldyia deyrolli (Vuillefroy 1864) is the best known, and is one of the reference points for the post-embryonic paternal care in insects (Ohba 2011).The life-cycle of these five species was supposed to be, in general, valid for other Lethocerinae including the old world members (Dudgeon 1999).Eggs of Lethocerus delpontei De Carlo, 1930 and Kirkaldyia deyrolli were described by Perez-Goodwyn et al. (2006: 155) and the authors assumed: "Due to the increase in size of the adults in order to have access to a vertebrate diet, the eggs should also increase in size."Aquatic mites (Arachnida: Acari: Hydracarina) were only accidentally noticed in association with Lethocerus spp.(Marlos 2010a) but regularly reported from other Nepomorpha (Proctor 1998;Hawking et al. 2009;Marlos 2010b).

MATErIAL ANd METHOdS
During field studies of benthic macro-invertebrate fauna the aquatic bugs were collected with the help of a 1mm mesh sized net by hand from the shallow zones of running and stagnant waters.Rivers and wetlands of the Gangetic plains were studied in Bihar, Jharkhand, West Bengal, New Delhi and Nepal and a total of 17 localities for Lethocerus species were discovered from 2004-2012 (Fig. 1).A few flying adults were also collected near light sources.All other records are nymphs and adults from aquatic habitats.The material was preserved together with all available benthic macro-invertebrates in a mixture of ethanol and formaldehyde for hardening the insects.After washing and sorting the specimens were kept in separate jars, labeled and transferred into 70% ethanol.The maximum body length and the width of all specimens was measured using a slide caliper.In adult specimens the distinction of males and females was based on the genital plate which was studied with a Leica stereo-zoom microscope.Attached pupae were studied by a high magnification Olympus microscope.
Identification of adults was done with the keys provided by Menke (1960) and Perez-Goodwyn (2006).The voucher material is deposited in the collection of the Zoological Survey of India, Gangetic Plains Regional Centre in Patna.All specimens deposited with their location and registration numbers are listed in the results.

rESuLTS
Two species of giant water bugs could be identified.Lethocerus spp. was found in eight lowland rivers and eight stagnant water bodies of the floodplains.The aquatic habitats of adults were predominantly large rivers but also one oxbow lake and one pond, whereas the exclusively aquatic nymphs (or larvae) were found only in temporary shallow ditches of riverine floodplains.These reproduction habitats are all characterized by seasonal water during the monsoon period starting from the month of June/July onwards.They dry completely after the following winter and are not inhabited by any fish fauna.The aquatic vertebrate fauna herein consist of tadpoles and adult amphibians which were abundantly found during the inundation period.

Biological observations
A total of 35 specimens of Lethocerus indicus were included in the present analysis (Image 1), these are in particular 22 nymphs, eight male and five female adults (Table 1).The nymphs can be grouped into four different instars according to their body size; the length is given here in mm.The first available development stage (1 st instar on Image 1) ranges from 8.5-9.3 mm and is represented by six individuals but approximately one dozen of the same sized nymphs were additionally observed in localities no. 12 and 13 at Hajipur.The next following instars reaching 13-19.5 mm (2 nd instar on Image 1, two nymphs), 30-33.1 mm (3 rd instar on Image 1, seven nymphs) and 47-52 mm (4 th instar in Image 2, seven nymphs) respectively.The presence of aquatic nymphs was observed only in the months of July and August.
The adults were identified males or females based on the different shape of the genital plates (Images 3,4).The male individuals reach 64-73 mm whereas the females are generally larger with 73-76.3mm body length.Submerged diving adults were observed in large and deep water bodies in the months of February, May, June, September and November.A single flying terrestrial adult was found in the month of August whereas they could be frequently seen in large numbers during the month of October, the monsoon not ending until the first week of November.
Three adult males and one female individual of the preserved material bear upto 7-47 ventrally attached pupae on the underside of the thorax and between the second and third pairs of legs.These are 'protelean parasites' with inactive pupa-like stages (Proctor 1998;Hawking et al. 2009) that represent the 'protonymph' and 'tritonymph' of aquatic mites (Acari: Cohort Parasitengona: Hydracarina) having striking similarity with eggs of Nepomorpha (Andersen & Weir 2004).The pupae of 1.0mm total length (Images 5,6) contain aquatic mites with four visible eye spots and readily developed legs.They have a yellowish-ochre color with white apices.The pupae-bearing male giant water bugs were found in the months May and June.A single female individual with only a few attached pupae from Damodar River was collected during floods in the month of September.Repeated observations of adults with ventrally attached pupae suggest that these are host-specific mites of Lethocerus spp.and they are not deposited accidentally using giant water bugs as substrate.
The single adult male specimen measured 55.2mm Image 2. Lethocerus patruelis, ventral side of adult male from locality no.17 with attached pupae of juvenile mites (Hydracarina).Sikkim was mentioned by Thirumalai (2007).
The adult male individual bears around 45 ventrally attached pupae of aquatic mites (Hydracarina) on the underside of the thorax between the second and third pairs of legs.The pupae of 1.0mm total length contain mites with four visible eye spots and readily developed legs (Image 7).They have a light yellow color with white apices.Thus the behavior of the mite's pupal stages resembles the observations which were made for Lethocerus indicus in the lower Gangetic plain.
The habitat is a pollution-free and clear lowland river that originates from the Gangetic plain of northern Bihar.The Budhi Gandak is characterized by a very

Prey
The subfamily Lethocerinae is known to be a vertebrate specialist that preys upon fish, amphibians, snakes and juvenile turtles.Lethocerus americanus (Leidy, 1847) is reported to prey also on aquatic Insecta, Cladocera and Gastropoda (Ohba 2011).

Nymphs
The species identity of nymphs is simply concluded from the adults found in the neighboring localities.It has to be mentioned that neither have their distinguishing characters been sufficiently studied nor have the wingless juvenile development stages of Lethocerus species been considered in the identification keys (Merritt & Cummins 1996;Goodwyn 2006;Huber et al. 2006).In the present study four instars of Lethocerus indicus were found in the field, also five moltings have been reported for North American species.The smallest nymphs of Lethocerus indicus which were found in the Gandak floodplain from the second half of July onwards already had a body length of 8.5-9 mm.

Habitat selection
It is noteworthy that the aquatic habitats of the adult Giant Water Bugs of the genus Lethocerus differ from the habitats of their nymphs which suggest a partly divided strategy in their life cycle.The instars of aquatic nymphs do not live in competition or under predatory pressure of fishes since they were found only in temporary water bodies.Thus the strategy to use such habitats for breeding shows the biological importance to protect the nymphs and to provide an easily capable food resource in the form of invertebrates and amphibians such as various sized tadpoles.In contrast, the adult winged stages inhabit permanent water bodies which harbor rich fish diversity.

Protelean 'Parasites'
These Hydracarina have a complex life-cycle, consisting of a parasitic larva, two inactive pupa-like stages that represent the protonymph and tritonymph, and active predatory deutonymphal and adult stages (Proctor 1998).The attached Hydracarina larvae are parasitic while deutonymphs and adults are predatory.Pupation takes place on the parasitized invertebrate.Our observations show that only adults of Lethocerus species in rivers are colonized by aquatic parasitengone mites.Out of these mostly the second stage (tritonymphs) have been found in the months of May and June (localities no. 2, 10, 11) whereas a few smaller protonymphs were available in the month of September (locality no.4).Both species Lethocerus indicus and Lethocerus patruelis bear remarkable similar tritonymphs which may represent the same species of Hydracarina.Although adult mites were not found in the present survey, the nymphs display unique characters that differ from previous observations (Marlos 2010a) as well as other Hydracarina of the study area.

Life cycle of Lethocerus species in the Gangetic plains
The first instars of aquatic nymphs appear in July and August in shallow temporary water bodies.They grow fast within two months and they molt several times, of which four instars of aquatic nymphs plus the winged adults have been observed.Adult dispersal by flying was seen frequently during the month of October till the first week of November.From winter to the month of June the adult Lethocerus species display an aquatic life style Image 7. Pupae of juvenile mites (Hydracarina) attached on male Lethocerus patruelis from locality no. 17.

Life history of giant water bugs
Nesemann & Sharma inside large water bodies like rivers, oxbow lakes and fishponds.During the aquatic period adult Lethocerus is host to parasitengone aquatic mites (Hydracarina) which spend a part of their life-cycle as parasitic "pupae" (protonymph, tritonymph) attached on adult bugs.Usually they are found in one to three groups attached on the ventral side of males.Tritonymphs of the second development stage of mites are carried from the first week of May to mid-June, smaller protonymphs in September.

Figure 1 .
Figure 1.Map of sampling sites in the Gangetic plains of India and Nepal .
frequency of the Lethocerus indicus population from the Gangetic plains.Four instar stages of nymphs plus adults were recorded in nature.

Image 5 .
Lethocerus indicus, ventral side of adult male from locality no. 9 with attached pupae of juvenile mites (Hydracarina).Image 6. Close up of the pupae of juvenile mites (Hydracarina) attached on male Lethocerus indicus from locality no. 9. Image 3. Male genital plate of L. indicus from locality no.10.Image 4. Female genital plate of L. indicus from locality no. 4. dense benthic molluscan fauna (five Bivalvia species, 11 Gastropoda species), the dragon fly nymph Macromia cf.moorei Selys, 1874 and accompanying adult bugs Laccotrephes griseus (Guerin-Meneville, 1844), Laccotrephes ruber (Linnaeus, 1764), Ranatra elongata Fabricius, 1790, Ranatra filiformis Fabricius, 1790.The Life history of giant water bugs Nesemann & Sharma total number of macro-invertebrate taxa reaches 25 species at the spot where the adult Lethocerus patruelis has been discovered.Thus water condition and biological community indicate high habitat quality in comparison with Lethocerus indicus habitats which have slightly turbid water and visibly higher eutrophication.

Table 1 . Sampling data and measurements of Lethocerus indicus preserved material.
was already noticed for New Delhi where both L. indicus and L. patruelis were identified by Perez Goodwyn(2006).Furthermore, the presence of L. patruelis in