International Journal of Microbiological Research 8 (1): 19-24, 2017 ISSN 2079-2093 © IDOSI Publications, 2017 DOI: 10.5829/idosi.ijmr.2017.19.24

Millipedes as Host for Microbes - A Review

Anbarasan Dhivya and Periasamy Alagesan

Post Graduate and Research Department of Zoology, Yadava College, Madurai, India

Abstract: This review mainly highlights the microbial load that abode in the gut of different millipede species. These diplopods are capable of digesting the leaf litter which they feed was only by the aid of microbial communities which have the ability to produce various hydrolytic enzymes that are highly responsible for the degradation of feed consumed by the millipedes. Millipedes are not well equipped for the degradation of the plant detritus, thereby maintaining a symbiotic relationship with bacteria, makes millipedes as effective decomposers. Reports are also available on the existence of microbial populations in the soil, leaf litter and the faecal pellets of millipedes. The microbes lodging in the gut may expel along with faeces, so some species act as coprophagy, which feed on their own faecal material. Interestingly, the journey of leaf litter through the intestinal wall, results in repopulation of feces with bacteria while reaching the hindgut. This review makes clear, that litter alone may not be the feed of millipedes instead, microorganisms may be the targeted food source which hires the litter as a vehicle for consumption of microbes.

Key words: Millipedes Gut microbes Decomposers Coprophagy

INTRODUCTION the decomposition processes of decaying plant material. The food source of the millipedes is the forest Millipedes are terrestrial arthropods and third diverse litter. The digestion of leaf litter and decaying matter is group after Insecta and Arachnida. They are commonly not only effected by millipedes but also due to the gut called as thousand legged animals. They bear two pair of microbiota, which play a vital role in converting them. The legs in each segment. Literally they are slow movers when gut of millipedes may represent a reservoir of bacterial compared to centipedes. They are sexually dimorphic. species. The secretion of amylases, cellulases, etc., by Similar to earthworms, they also involve in the the millipede gut-wall cells,the intestinal microbial humification and nutrient enrichment of soil. They lack a community plays a significant role in the breakdown of waxy cuticle, so they spend most of their time in moist the plant polymers[2]. The present review delivers a clear, humid environment and they dwell under the top soil. recent and concurrent idea that millipedes alone do not They are efficient burrowers which are confined to degrade the plant litter but the bacterial population monsoon and post monsoon season. Millipedes use their residing in their intestine promotes the animals as efficient sensory organs to measure the humidity. The diplopods detrivorous arthropods. undergo moulting and have the habit of feeding on its own exuvia in order to obtain the calcium content from it. Morphology and General Characteristics: Millipedes are These species exhibit a protective mechanism against its heliophobic that are sensitive to direct sunlight. The head predators such as the hard exoskeleton, coiling of its of the millipede is rounded above and flattened below. body and formation of ball, release of offensive chemicals The first three segments are diplosegments formed by the such as alkaloids, benzoquinones, phenols, terpenoids fusion of two embryonic segments which contains two and hydrogen cyanidethat are bad odoured and can cause pair of spiracles that opens into an internal pouch and irritation to the predators like itching, pain, local erythema, connects to the trachea through which they breathe. edema, blisters, eczema and occasionally cracked skin [1]. Heart runs along the entire length of the body and aorta They are representatives of the soil macrofauna that live stretching into the head. Two pairs of malpighian tubules in the deciduous forest litter and play an important role in are present at the mid part of the gut which functions as

Corresponding Author: PerisamyAlagesan, Post Graduate and Research Department of Zoology, Yadava College, Madurai, India. E-mail: [email protected]. 19 Intl. J. Microbiol. Res., 8 (1): 19-24, 2017 the excretory organ. The sex organs are referred as on earthworms [11]. Coprophagy is quite common in this gonophores and vulvae in the male and the female animalgroup, including the consumption of their own respectively [3, 4]. pellets and exuvia [12]. Some species are coprophagy and A unique report reveals that some species exhibits a mycophagy. While feeding, the millipedes tend to move parthenogenetic reproduction especially in Poratia vertically and put their anterior body into the soil and salvator. Frequently the thelytoky group undergoes simultaneously they leave their faecal egestion on the soil parthenogenesis for their reproduction from which only surface. females are hatched from the unfertilized female independent from males. These females are resistant Gut Microbiota: Interaction between millipedes and against desiccation when compared to male that undergo microorganisms are very important aspect of the bisexual reproduction. They viably produce their next decomposition process. It is known, for example, that the generation easily because they do not suffer the risk of comminution of plant material by millipedes as well as by desiccation, predation and spend less energy. All these other soil feeding animals increases the surface area were possible because of lack of copulation. available for the microbial colonization [13] and the Parthenogenesis in this particular species, P. salvator is intestine of these animals act as favourable environment affected by the presence of Flavobacterium [5]. This kind for the bacterial growth [14,15]. The presence of gut of reproduction takes place in the population having small bacteria in the millipede is also a possible source of the number of male millipedes than females or even at a total amino acid detected in the fecal pellets due to the absence of male (Spanandry). excretion of gut lining and associated symbiotic microorganism. The degradation of glucose, Millipedes Distribution: The marine littoral, freshwater predominantly sourced from cellulose suggests the habitats, deserts, zonal tundra, high mountains, caves, presence of appreciable cellulolytic activity in the gut of deeper soil, epiphytes, the bark of trees, tree canopies, the millipede associated with either endogenous most of such difficult environments are only marginally cellulases or symbiotic gut bacteria capable of cellulose populated by diplopods, but caves and high altitudes are degradation. often full of them. The oldest fossil millipedes, apparently It is estimated that the millipedes assimilate only one detritivores, are known from themid-Silurian and tenth from its total feed intake and 90% of the leaf litter Devonian, while since the carboniferous they became fed were egested out as fecal pellet. Millipedes transform quite diverse [6]. Some species belong to marine littorals, plant material into fecal pellets that affect important Polydesmus denticulatus (Polydesmida: Polydesmidae) physiochemical properties, specifically by decreasing the can stay alive underwater for 75 days [7]. Millipedes carbon/ nitrogen ratio and in the subsequent process of lacking any morphological or physiological adaptations decomposition [16-18]. Millipedes are literally not able to go for vertical migrations along the tree trunks to survive digest the leaf litter or feed intake on the whole and are [8]. Deserticulous millipedes are also available [9]. It is not well equipped with specialized enzymes which enables reported that the abundance of millipede population is to digest, so they are enhanced by the microbial found in the Alagar hills, Madurai [10] and Elagiri districts population that accommodate in the gut of the animals. of Tamilnadu. It reveals that the diversity index is high at The food intake are grained by the mandibles and then the mid elevation. The season, temperature, humidity and subjected to enzyme action from the salivary glands, mid altitude are the important parameters for the availability of gut epithelium and finally the most important role played millipede species. Millipedes are considered as the bio- by the microbial community in the digestive track of the indicators of the environment, because their rich millipede. It is suspected that micro- organisms in the appearance and activity during the rainy season and alimentary canal play a crucial role in the digestion of food humid conditions whereas the increase in temperature and and indirectly influence the fluxes of nutrients [3, 19]. lack of rainfall results in decrease of millipedes. The transformation of leaf litter into faeces by saprophagous macroarthropods can influence the Feeding Biology: Millipedes are estimated to consume decomposition process in several ways. It has been 10–15% of the annual leaf fall in temperate forest. shown that the chemical composition of the leaf material Detritivores predominating, only a few species can be is changed during gut passage [20]. Bacterial count in the qualified as omnivores, even fewer as carnivores feeding gut of millipede species is presented in Table 1.

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Table 1: Bacterial count in the gut of millipede species. Cylindroiulus caeruleocinctus is a habitat for Bacterial count Citrobacter freundii, Pantoea agglomerans, Serratia Millipede Species (CFU/g dry wt.) Author marcescens, Raoultella planticola and Salmonella Jonespeltis splendidus 153.5x104 Bano et al. [21] Glomeris marginata 23.4x107 Anderson and Bignell [22] arizonae [28]. The following bacterial species such as C. G. marginata 22.8x108 Ineson and Anderson [23] freundii, P. agglomerans, R. planticola and Ommatoiulus sabulosus 3.84x106 Jarosz and Kania [24] Xanthomonas maltophilia are found in the gut of the 8 Xenobolus carnifex 1.4x10 Alagesan et al. [25] millipede Ommatoiulus sabulosus [28]. Arthrosphaera magna 3.8x1011 Ramanathan [2] Aulocobolus newtoni 3.1x1011 Ramanathan [2] Bacterial counts showed that bacterial growth was Spinotarsus colosseus 4.7x1011 Ramanathan [2] enhanced both in the guts and faeces of these animals as a habitat for soil bacteria has received little attention. The total heterotrophic bacteria from three different The feeding activities of soil invertebrates can cause millipede species namely Arthrosphaera magna, gross shifts from fungal to bacterial activity in litter and Aulocobolus newtoni and Spinotarsus colosseus from soils [14, 22]. Bacillus thuringiensis was one among the Alagar hills of Tamil Nadu were characterized [26]. It is microflora isolated from the gut of A. newtoni [29]. It is recorded that the species A. magna with pH of 6.4, the Gram positive bacteria which produce Cry proteins on colony forming unit and enzyme producing bacteria are sporulation. The count of B. thuringiensis was higher in amylase(Alcaligenes faecalis) 1.6x1010 , protease faeces for first three days when compared to ingested (Enterobacter aerogenes) 1.4x108 , cellulase food. Then after 9days cell count of B. thuringiensis were (Corynebacterium sp.) 2.0 x 1010 , chitinase (Streptococcus same in both leaf litter fed by the millipede and expelled mitis) 3.7x1010 , xylanase(Micrococcus varians) 4.0x10 .8 faecal material. When known amount of B. thuringiensis The bacterial species found in the gut are A. faecalis, was inoculated in the hindgut there was a considerable Corynebacterium sp., S. mitis, M. varians and E. increase in cell count upto 10 fold within 5 to 24 hours, aerogenes. whereas when it was incubated with midgut fluid, there The species A. newtoni recorded a pH range of 6.7, was a drastic change which resulted in 10 fold decrease the colony forming unit and enzyme producing bacteria after 5hours. The outcome of this is that, the are amylase (Citrobacter freundii) 1.2x1010 , protease multiplication of B. thuringiensis actively takes place in (Bacillus cereus) 1.0x108 , cellulose (Corynebacterium the hindgut but lower in midgut and foregut of litter kutcheri) 4.3x1010 , chitinase (Lactobacillus fermentum) feeding animals. This is because of the antimicrobial 1.9x1010 , xylanase (Corynebacterium xerosis) 5.7x108 . activity of midgut fluid. The bacterial species found in the gut are C. freundii, The strains of bacteria and fungi were preliminarily C.kutcheri, L. fermentum, C. xerosis and B. cereus [26]. identified according to cultural, morphological, The species S. colosseus recorded a pH range of 6.3, physiological and biochemical properties to genera or the colony forming unit and enzyme producing bacteria species level from the gut isolate of millipede. Complex are amylase(Bacillus pumilis) 1.9x1010 , protease (Bacillus microbial communities of millipede composed of many subtilis) 1.8x108 , cellulose (Pseudomonas aeruginosa) species of partly true soil-bacteria Bacillus, Micrococcus, 3.6x1010 , chitinase (Micrococcus roseus) 2.9x1010 , xylanase Clostridium, Corynebacterium, etc. and partly typical (Lactobacillus casei) 5.0x108 . The bacterial species found intestinal ones Klebsiella, Enterobacter, etc. [27] as well in the gut are B. pumilis, P. aeruginosa, M. roseus, L. as actinomycetes and fungi [30] colonize the mid- and casei and B.subtilis [26]. hindgut contents of millipedes. It is said that the Similarly, the species Schizophyllum sabulosum is microflora in the litter gets multiplied in the gut of the litter the host for the following bacterial community genera of feeding animals and the number of these microbes in the Klebsiella, Sarcina, Bacillus and Corynebacterium [27]. faeces is higher than in the ingested food [22]. The millipede, Glomeris marginata contains the bacterial The mid gut of millipede is the main site for population like Pseudomonas alcaligenes, Klebsiella degradation of cellulose and hemicelluloses and the pneumoniae [23]. The species Ommatoiulus sabulosus amount of pectin degradation in hind gut is considerably contains Escherichia coli, Enterobacter aggomerans, high. Millipedes distributed among different habitats are Klebsiella, Pseudomonas fluorescence, Sarcina [24]. known to harbor more or less similar gut microbial The bacterial community Proteus mirabilis and community. Microbial population was differentially altered Citrobacter freundii are the dwelling place in the gut of from food to gut to fecal pellets amongst the the millipede Xenobolus carnifex [25]. The gut of millipede Arthrosphaera species. The population of aerobic

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heterotrophic bacteria was highest. They steadily leaf litter or soil [39]. The fecal pellets of the species A. increased from leaf litter to gut content and leaf litter to magna contains several bacterial groups like alpha- feces. The population of rhizobia was next to proteobacteria that includes Methylobacterium populi, heterotrophic bacteria. Third highest population was Novosphingobium subarcticum, beta-proteobacteria actinomycetes. The least among the bacteria studied was includes Cupriavidus basilensis, pseudomonas putida, phosphate solubilizing bacteria and its dynamics was gamma-proteobacteria includes Citrobacter similar to rhizobia and actinomycetes. Filamentous fungi amalonaticus, Klebsiella pneumonia, Pantoea were higher than the yeasts and their population agglomerans, Pseudoxanthomonas koreensis and bacilli substantially decreased from leaf litter to feces. includes Bacillus arsenicus, Bacillus licheniformis and The hindgut consists of bacteria, actinomycetes and Bacillus subtilis [40]. fungi and maximum bacterial population was found in It is also reported that there is methanogenic bacterial feces as proliferation of bacteria takes place in the hindgut diversity in the digestive track of millipedes. The species [31]. Increase of bacterial population in feed to fecal that emitted methane are mostly found in the juliform pellets ranged between 10 and 100 folds, whereas fungal Julida, Spirobolida, Spirostreptida [41] Methane is known population decreased up to 3.5 folds. There is a view that to be produced by almost all tropical millipedes but is the gut passage of leaf litter to fecal pellets increases the generally not to be produced by millipedes and bacterial population, while decreases the fungal cockroaches from temperate climates [42]. However, population [22]. Nearly 50% of millipedes preferred detected methane production in several species of feeding ascospore-releasing structures of fungi [32] for European millipedes mainly Julida, while other groups the assimilation of calcium by diplopods. Diplopods Polydesmida and Glomerida do not [43]. assimilate high calcium content and transform into calcium oxalate to form calciferous tergites [33] this is the reason CONCLUSIONS behind the decrease in fungal population. The animals

while consume fungi, the rate of co2 waste was high, while The conditions of millipedes gut like high humidity the assimilation efficiency had its increasing level when and low oxygen are creating a suitable habitat for the bacteria are ingested [34]. anaerobic bacteria to grow. The association between the The millipede Pachyiulus flavipes serves as a millipedes and gut flora are always mutualistic, bacteria by reservoir for the bacteria like Pseudomonas putida and the production of certain enzymes make it available for the Pseudomonas stutzeri, which thereby show their high millipedes for its survival even in crucial environments population density in the fecal pellets when compared and inturn the millipedes provide an environment that is with their gut [35]. There seems to be a selective suitable for the bacteria to dwell such as regulated assimilation and elimination of microbes passing through temperature, humidity, pH as a symbiotic relationship. the gut of invertebrates [36]. Communities of microbes present on faeces can be a The four bacterial communities namely heterotrophic source of nutrition for millipedes or otherwise it can be a bacteria, actinomycetes, phosphate solubilizing bacteria source of various enzymes which the millipedes are not and rhizobia that are ascending from leaf litter to gut able to produce by themselves. The presence of gut content and finally has a greater load in the feces [36, 37]. bacteria in the millipede is also a possible source of amino But the filamentous fungi and yeast population tends to acids detected in the faecal pellets due to the excretion of decrease when compared to bacterial community. The gut lining and associated symbiotic microorganisms. The fungal population plays a vital role which make the bacteria in the foregut were probably present as a result availability of essential amino acids, vitamins etc., to the of ingestion and are therefore not part of the resident millipedes which could not be gained by them in any other flora. During winter, the number of foregut bacteria varies way especially for the desert dwelling species. This was greatly from individual to individual, whereas the midgut reported in the pill millipede species A. magna [37, 38]. and hindgut of all individuals retain large populations The However, the gut of these pill millipedes are devoid of forest ecosystem’s most essential and important lignocelluloses degrading bacteria and they fully depend component is millipede, which is a predominant organism only on the soil borne microbe and leaf litter bacteria for playing a major role in enriching soil fertility, recycling of the degradation or breakdown of lignocellulose into plant material in ecosystem in relation with gut assimilable form for the animals[35, 38] and several bacteria.Millipedes change the chemical composition of species are unique to their gut that are not native to the the leaf material during ingestion and favour the

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