New leptestherid clam shrimps (Pancrustacea: Branchiopoda: Spinicaudata: Leptestheriidae) from peninsular India.

Spinicaudatan clam shrimps are an evolutionarily ancient lineage restricted to temporary freshwater pools. Use of classical morpho-taxonomic approaches alone have led to some issues in the taxonomy of this group, which are now being resolved through integrative taxonomy. Here, we describe two new leptestherid spiny clam shrimps Leptestheria chalukyae sp. nov. and Leptestheria gomantaki sp. nov. from peninsular India based on their unique morphological characters and distinct phylogenetic position. We also re-describe Leptestheria nobilis and present an overview of the morphological characters of all the Indian leptestherids. Most of the conventional taxonomic characters appear to overlap among all the Indian species, although the combination of occipital condyle shape and the cercopod marginal spines arrangement in combination, appear to be useful in separating leptestherid species.

Assigning the Australian clam shrimp Eoleptestheria (Branchiopoda: Spinicaudata: Leptestheriidae) to a new species status.

We assign the only Australian leptestherid clam shrimp, Eoleptestheria ticinensis (Balsamo-Crivelli, 1859) to a new species status, Leptestheria timmsi sp. nov., based on the latest molecular phylogeny of spinicaudatan clam shrimps from a previous study. We provide a redescription of the species, highlighting morphological details not explored previously for this species. We also present a comparative account of all Eoleptestheria species and populations throughout the world. The head morphology of Leptestheria timmsi sp. nov. overlaps with other populations of Eoleptestheria and a few Leptestheria species. The telsonic and cercopod marginal spines seem to increase in size posteriorly in L. timmsi sp. nov., unlike in many other Eoleptestheria populations.

Phylogeny and Biogeography of Spinicaudata (Crustacea: Branchiopoda).

Spinicaudata (spiny clam shrimp) is a taxon of Branchiopoda occurring since the Devonian and today it occurs nearly globally in temporary water bodies. We present the most species-rich phylogenetic analyses of this taxon based on four molecular loci: COI, 16S rRNA, EF1α and 28S rRNA. Our results support previous findings that Cyzicidae sensu lato is paraphyletic. To render Cyzicidae monophyletic we establish a fourth extant spinicaudatan family to accommodate Eocyzicus. Within Cyzicidae, none of the genera Cyzicus, Caenestheria or Caenestheriella are monophyletic, and the morphological characters used to define these genera (condyle length and rostrum shape) are not associated with well-delimited clades within Cyzicidae. There is insufficient resolution to elucidate the relationships within Leptestheriidae. However, there is sufficient evidence to show that the leptestheriid genera Eoleptestheria and Leptestheria are non-monophyletic, and there is no support for the genus Leptestheriella. Molecular clock analyses suggest that the wide geographic distribution of many spinicaudatan taxa across multiple continents is largely based on vicariance associated with the break-up of Pangea and Gondwana. Trans-oceanic dispersal has occurred in some taxa (e.g., Eulimnadia and within Leptestheriidae) but has been relatively rare. Our results highlight the need to revise the taxonomy of Cyzicidae and Leptestheriidae and provide evidence that the global spinicaudatan diversity may be underestimated due to the presence of numerous cryptic species. We establish Eocyzicidae fam. nov. to accommodate the genus Eocyzicus. Consequently, Cyzicidae comprises only two genera -Cyzicus and Ozestheria. Ozestheria occurs also in Africa and Asia and Ozestheria pilosa new comb. is assigned to this genus.

Review of the Eulimnadia (Branchiopoda: Spinicaudata: Limnadiidae) of Tropical Asia, with the Description of a New Species.

We describe a new species of Eulimnadia from the Oriental region using museum specimens collected from India and fresh material from Thailand. This species has egg morphology resembling E. magdalensis s. lat. and E. chaperi, but distinctly differs from them by presence of a narrow depression at the polygon floor. We also comment on the species status of E. khoratensis from Thailand based on egg morphology, and present a taxonomic key for identification of tropical Asian species of Eulimnadia.

Taxonomical account of the Indian Cyzicid Ozestheria annandalei (Daday, 1913) (Branchiopoda: Spinicaudata) from peninsular India.

The spinicaudatan family Cyzicidae represents a group of morphologically variable species whose taxonomy is still being updated (Schwentner et al. 2009, 2015, 2020; Rogers et al. 2017). There are 21 species of spinicaudatans currently reported from the Indian subcontinent out of which 4 species represent the Cyzicidae family (sensu Rogers Padhye, 2015; not considering the species from Eocyzicus genus which are now shifted to Eocyzicidae family sensu Schwentner et al. 2020). Ozestheria indica (Daday, 1913), one of the four species, has been re-described very recently (Padhye, 2020). Ozestheria indica and Ozestheria annandalei (Daday, 1913) are the two valid species currently known from peninsular India (Padhye, 2020; Rogers, 2020 2020). Padhye et al., (2015) re-described the female of a Cyzicus sp. which was later confirmed as C. annandalei (Rogers Padhye, 2015).

Taxonomical account of the Indian Cyzicid Ozestheria annandalei (Daday, 1913) (Branchiopoda: Spinicaudata) from peninsular India.

The spinicaudatan family Cyzicidae represents a group of morphologically variable species whose taxonomy is still being updated (Schwentner et al. 2009, 2015, 2020; Rogers et al. 2017). There are 21 species of spinicaudatans currently reported from the Indian subcontinent out of which 4 species represent the Cyzicidae family (sensu Rogers Padhye, 2015; not considering the species from Eocyzicus genus which are now shifted to Eocyzicidae family sensu Schwentner et al. 2020). Ozestheria indica (Daday, 1913), one of the four species, has been re-described very recently (Padhye, 2020). Ozestheria indica and Ozestheria annandalei (Daday, 1913) are the two valid species currently known from peninsular India (Padhye, 2020; Rogers, 2020). Padhye et al., (2015) re-described the female of a Cyzicus sp. which was later confirmed as C. annandalei (Rogers Padhye, 2015).

First record of Magnospina Sousa, Elmoor-Loureiro amp; Santos, 2016 (Cladocera: Chydoridae: Aloninae) from tropical India.

David Frey raised the hypothesis of non-cosmopolitanism in Cladocera in a series of his publications (Frey 1982, 1987). Since then, researchers have taken the effort to revise species groups with wide geographic distribution around the world (e.g. Kotov Dumont 2000; Sinev 2015; Van Damme Dumont 2008a; Sinev Elmoor-Loureiro 2009). The polyphyletic nature of the genus Alona Baird, 1843 (Van Damme et al. 2010) has further substantiated the view that cladocerans are non-cosmopolitan, with the creation of many new genera to accommodate the several species groups known within this genus (Van Damme Dumont 2008b; Sinev Kobayashi 2012; Van Damme Dumont 2009; Alonso Sinev 2019).

Taxonomical account of the Indian spiny clam shrimps (Crustacea: Branchiopoda: Spinicaudata) from the Smithsonian National Museum of Natural History, USA.

I present taxonomical re-descriptions of two Indian spinicaudatan species deposited at the Smithsonian National Museum of Natural History, USA, emphasizing on a few characters not highlighted in the original descriptions. Specimens collected from South India and deposited in the museum forty years ago were assessed and re-described. The study shows that the Cyzicidae species deposited as Caenestheriella sp. is Ozestheria indica while the Eulimnadia species is the widely distributed Eulimnadia michaeli. Many of the taxonomical characters were variable and did not exactly match the earlier description for both the species. Ozestheria indica could be distinguished from other valid Indian species on the telson characters and its occurrence proximity to the type locality of the species. This species is currently restricted to Peninsular India. Eulimnadia michaeli could be identified based on its characteristic egg surface morphology, though, the inner layers of the egg did vary when compared with the western Indian population. Using museum material, the validated species tally of Indian spiny clam shrimps now stands at 8 while 15 still await re-examination.

A new species of genus Eulimnadia Packard, 1874 (Branchiopoda: Spinicaudata: Limnadiidae) from India with an updated key for some Indian species.

We describe a new species of Eulimnadia, Eulimnadia bondi sp. nov., from the state of Goa, India. This is the 8th species to be described from the country and first from this state. This species differs from all other Indian species in having unique egg morphology-cylindrical egg with dilated borders at both ends and a specific internal eggshell structure. Specimens showed intra-population morphological variation as observed in other species from the genus. Eulimnadia bondi sp. nov. egg resembles the South American E. colombiensis and the Australian E. australiensis. These species are however distinguished on egg morphological details and by some morphological characteristic of adults. Based on the available information, we present an overview of the distribution of the different Indian Eulimnadia species and an updated identification key based on egg morphology.

An updated and detailed taxonomical account of the large Branchiopoda (Crustacea: Branchiopoda: Anostraca, Notostraca, Spinicaudata) from the Yale North India Expedition deposited in the Yale Peabody Natural History Museum.

We present an updated taxonomic account of a few of the large branchiopod species present in the collections of the Yale Peabody Museum of Natural History. Our study shows that the Artemia species in the collections is not Artemia salina given the presence of spines at the base of the penis and its frontal knob morphology. This population cannot be assigned to any particular species due to lack of comparative material of other Artemia species and therefore, now reduces the authentic distribution record of Artemia salina to just one in the subcontinent. The morphology of eggs taken from the Streptocephalus longimanus allotype is distinct from what has been described later by having lower number of polygons on the egg surface. The gross morphology of the Triops cancriformis from Punjab, Pakistan resembles those of the Kashmir population from India except for its larger size. Based on the morphological comparison, we show that Eocyzicus deterrana is indeed synonymous to E. hutchinsoni. This work further changes the total species tally of large branchiopods from 44 of the Indian subcontinent to 45.

Molecular phylogenetic investigations of Triops granarius (Lucas, 1864 (Branchiopoda: Notostraca) from the type locality of the former Apus orientalis Tiwari, 1952 and three other localities in the Western Ghats of India.

We investigated the phylogenetic position of Triops granarius populations from four localities in the Western Ghats using partial sequences of three mitochondrial genes (COI, 12S rRNA and 16S rRNA) publicly available on the GenBank database. One of these localities, Panchgani, is particularly important since it is the type locality of the former Apus orientalis which is currently treated as a junior synonym of T. granarius. Phylogenetic analyses reveal that populations from all the four localities (Kolhapur, Chalkewadi, Panchgani, and Dighi) form a single lineage, which is here named 'Maharashtra lineage'. One of the two previously published samples from India, treated as lineage 'Triops granarius 4' is nested within this clade. The 'Maharashtra lineage' is separated from other lineages by mean maximum likelihood distance ≥ 11.9% in the COI gene. This distance is suggestive of a separation on species level from other lineages of T. granarius. This interpretation is further supported by a conservative genus-wide species delimitation analysis performed in the present study upon application of the Automatic Barcode Gap Discovery method. The 'Maharashtra lineage' branches out in two sub-lineages of Panchgani+Kolhapur and Dighi+L4+Chalkewadi samples, separated by 5.9% mean ML distance (uncorrected p-distance = 5.4%) in COI. The application of a 5% threshold to the COI dataset would thus even suggest a possible differentiation of both sub-lineages on species level. Comparative morphological data is presently not available because most vouchers associated with the sequences were depleted for DNA extraction. Further studies are needed in order to prepare a sound taxonomic revision. Thus, in the current study we refrain from re-instating Apus orientalis to full species status (likewise, for other names of Asian taxa in this morphogroup, including Apus sinensis Uéno, we retain the status as junior synonym of T. granarius). Nonetheless, our study highlights the fact that still there may be undescribed cryptic species associated with the specific name in this part of Western Ghats (Linnean Shortfall) and paves the way for future taxonomic investigations and conservation strategies for the genus Triops in India.

A new Indian record and morphological variation for Eulimnadia khoratensis Rogers et al., 2016 (Crustacea: Branchiopoda: Spinicaudata).

Eulimnadia Packard, 1874 is the most species rich and widely distributed genus within the Limnadiidae (Reed et al. 2015). Species identification relies primarily on egg morphology as adult morphological characters show high variability, sometimes within the same populations and hence of little use (Rabet, 2010; Rogers et al. 2012). Seven species are currently known from the Indian subcontinent (sensu Rogers & Padhye 2015) and SE Asia (Rogers et al. 2016). Of these, four species, viz. E. compressa (Baird, 1860), E. michaeli Nayar & Nair, 1968 and E. indocylindrova Durga Prasad and Simhachalam, 2004, E. azisi Babu and Nandan, 2010 are known from peninsular India (Padhye et al. 2015; Rogers & Padhye, 2015), although, much of this region remains unstudied (Padhye pers. obs.). With this background, we present a new Indian record of E. khoratensis Rogers, Dadseepai and Sanoamuang, 2016 from the Western region of Maharashtra state, India, extending its distribution a few thousand kilometers westwards.

Ontogenetic trajectory and allometry of Diplonychus rusticus (Fabricius), an Oriental aquatic bug (Hemiptera: Belostomatidae) from the Western Ghats of India.

Despite being one of the dominant groups in freshwater ecosystems, morphological and ontogenetic studies on aquatic Hemiptera have received little attention in the Oriental region. We present the ontogenetic trajectory and allometry of the widespread Oriental belostomatid species, Diplonychus rusticus (Fabricius) for the first time. We have measured nine different morphological variables throughout the growth of the bug using both field captured and laboratory reared specimens. Our results suggest that the developmental instars can be distinguished by the size variables, as seen in the Principal Component Analysis. On the basis of a CHAID (Chi-squared Automatic Interaction Detection) based regression tree, we also show that the characters - total length without head and maximum width - prove to be adequate for effective instar identification. The multivariate allometric growth pattern shows that different body parts exhibit different types of allometry. This is apparent in the allometry exhibited by forelegs and mid and hind legs, which show allometry of opposite polarities. This may be due to the different functions attributed to these body parts. Our results show that the growth pattern in D. rusticus is comparable with the New World genus Belostoma, suggesting a conserved growth pattern in the family Belostomatidae.

A new species of Leptestheria (Crustacea: Branchiopoda: Spinicaudata) from Western Maharashtra, India.

We describe a new species of Leptestheria from a rock pool in the Western Ghats of Maharashtra, India. This species is distinguished from all other Indian congeners by its distinct telson characters and occipital condyle. We also present a comparative table of useful morphological characters of all described Indian Leptestheria species.

Large branchiopod (Crustacea: Branchiopoda) egg morphology of Western Ghats, Maharashtra, India.

The eggs of many large branchiopods have taxonomic value and are commonly used as traits in species and/or generic descriptions. In this paper we present detailed descriptions and SEMs of resting eggs of seven of the eight species of large branchiopods found in the Western Ghats of Maharashtra, India. We highlight the inter- and intrapopulation egg morphological variation in Streptocephalus.

Note on Kurzia (Kurzia) latissima (Kurz, 1875) (Branchiopoda: Anomopoda: Chydoridae) from India.

Members of the chydorid genus Kurzia Dybowski & Grochowski, 1894 (Branchiopoda: Anomopoda) are typically known from the Holarctic and the Neotropics (Rajapaska & Fernando 1986; Hudec 2000; Elmoor-Loureiro 2002; Kotov, 2004). One representative of the sub genus Kurzia, Kurzia (Kurzia) latissima (Kurz 1875) has been reported from India, in the Oriental Zone. These reports either have unreliable or no illustrations (Rane, 1983; Michael & Sharma 1988; Padhye & Dumont 2015). Recently, Chatterjee et al. (2013) have stated that records of K. latissima from India appear morphologically similar to K. latissima s.str. but have not elaborated on it. The aim of this correspondence is to present an initial diagnosis and provide illustrations of a Kurzia latissima-like population from the Northern Western Ghats, Maharashtra Province, India, along with comments on its distribution.

Cholesterol anchored arabinogalactan for asialoglycoprotein receptor targeting: synthesis, characterization, and proof of concept of hepatospecific delivery.

Asialoglycoprotein receptors (ASGPR) are hepatocyte bound receptors, which exhibit receptor mediated endocytosis (RME) for galactose specific moieties. Arabinogalactan (AG), a liver specific high galactose containing branched polysaccharide was hydrophobized using cholesterol (CHOL) as a lipid anchor via a two step reaction process to yield the novel polysaccharide lipid conjugated ligand (CHOL-AL-AG). CHOL-AL-AG was characterized by Fourier transform infra red (FTIR) spectroscopy, (1)H and (13)C nuclear magnetic spectroscopy (NMR), size exclusion chromatography (SEC) and differential scanning calorimetry (DSC). Conventional liposomes (CL) and surface modified liposomes (SML) containing CHOL-AL-AG were prepared using reverse phase evaporation technique. Effect of CHOL-AL-AG concentration on particle size and zeta potential of SML was evaluated. Surface morphology of CL and SML was studied using cryo-transmission electron microscopy (cryo-TEM). In vitro binding affinity of SML and CL was evaluated using Ricinus communis agglutinin (RCA) assay. Cellular uptake of SML and CL was determined on ASGPR expressing HepG2 cell lines by confocal laser scanning microscopy technique (CLSM). FTIR spectra revealed bands at 1736 cm(-1) and 1664 cm(-1) corresponding to ester and carbamate functional groups, respectively. Signals at δ 0.5-2.5 corresponding to the cholestene ring and δ 3-5.5 corresponding to the carbohydrate backbone were observed in (1)H NMR spectrum of the product. CHOL-AL-AG possessed a mean average molecular weight of 27 KDa as determined by size exclusion chromatography. An endothermic peak at 207 °C was observed in the DSC thermogram of CHOL-AL-AG, which was not observed in thermograms of reactants and intermediate product. Synthesized CHOL-AL-AG was successfully incorporated in liposomes to yield SML. Both CL and SML possessed a mean particle size of ∼ 200 nm with polydispersity index of ∼ 0.25. The zeta potential of CLs was observed to be -17 mV whereas zeta potential of SMLs varied from -18 to -22 mV. RCA assay revealed enhanced binding of SML compared to CL confirming presence of galactose on surface of SML. CLSM studies demonstrated enhanced cellular uptake of SMLs compared to CL by HepG2 cells post 3 h administration indicating enhanced uptake by the ASGPR. Thus surface modified liposomes specific to target heptocytes demonstrate a promising approach for targeted drug delivery in liver cancer therapeutics.

First faunal inventory of large branchiopods (Crustacea: Branchiopoda) of Western Maharashtra, India with taxonomical and distributional comments.

An updated inventory of large branchiopods of the Western Maharashtra state, India is presented for the first time. Employing current identification keys, we have detailed the egg morphology of Eulimnadia indocylindrova, which shares some similarities with E. geayi from South America and E. alluaudi from Madagascar, based on these egg characters. A thorough re-description of few morphological traits of the same species is also presented. We also have described the female of a Cyzicus species seen rarely from the studied region. Distributional comments on few species are also reported.

Liposomes for targeting hepatocellular carcinoma: use of conjugated arabinogalactan as targeting ligand.

Present study investigates the potential of chemically modified (Shah et al., 2013) palmitoylated arabinogalactan (PAG) in guiding liposomal delivery system and targeting asialoglycoprotein receptors (ASGPR) which are expressed in hepatocellular carcinoma (HCC). PAG was incorporated in liposomes during preparation and doxorubicin hydrochloride was actively loaded in preformed liposomes with and without PAG. The liposomal systems with or without PAG were evaluated for in vitro release, in vitro cytotoxicity, in vitro cell uptake on ASGPR(+) cells, in vivo pharmacokinetic study, in vivo biodistribution study, and in vivo efficacy study in immunocompromised mice. The particle size for all the liposomal systems was below 200 nm with a negative zeta potential. Doxorubicin loaded PAG liposomes released significantly higher amount of doxorubicin at pH 5.5 as compared to pH 7.4, providing advantage for targeted tumor therapy. Doxorubicin in PAG liposomes showed superior cytotoxicity on ASGPR(+) HepG2 cells as compared to ASGPR(-), MCF7, A549, and HT29 cells. Superior uptake of doxorubicin loaded PAG liposomes as compared to doxorubicin loaded conventional liposomes was evident in confocal microscopy studies. Higher AUC in pharmacokinetic study and higher deposition in liver was observed for PAG liposomes compared to conventional liposomes. Significantly higher tumor suppression was noted in immunocompromised mice for mice treated with PAG liposomes as compared to the conventional liposomes. Targeting ability and superior activity of PAG liposomes is established pre-clinically suggesting potential of targeted delivery system for improved treatment of HCC.