Robert Price Higgins-Aspects of his life and his contributions to meiobenthic research (1932-2022).

The scientific life of Robert P. Higgins was devoted to meiofauna, microscopically small animals living in aquatic sediments from the intertidal to hadal depths worldwide. He focused on the taxonomy, life-history, and ecology of the marine taxa Kinorhyncha, Tardigrada, and Priapulida and co-discovered the phylum Loricifera. He improved the methods for studying meiofauna and contributed significantly to spreading knowledge about these animals. Aspects of his life are described and lists of his publications, taxa described, contributions to science, and honours received are provided.

Sediment-contact fish embryo toxicity assay with Danio rerio to assess particle-bound pollutants in the Tietê River Basin (São Paulo, Brazil).

The Tietê River and its tributary Pinheiros River receive a highly complex organic and inorganic pollutants load from sanitary sewage and industrial sources, as well as agricultural and agroindustrial activities. The aim of the present study was to evaluate the embryotoxic and teratogenic effects of sediments from selected locations in the Tietê River Basin by means of the sediment contact embryo toxicity assay with Danio rerio, in order to provide a comprehensive and realistic insight into the bioavailable hazard potential of these sediment samples. Lethal and sub-lethal effects were recorded, and high embryo toxicity could be found in the samples not only in the vicinity of the megacity São Paulo (Billings reservoir and Pinheiros River samples), but also downstream (in the reservoirs Barra Bonita, Promissão and Três Irmãos). Results confirm that most toxicity is due to the discharges of the metropolitan area of São Paulo. However, they also indicate additional sources of pollutants along the river course, probably from industrial, agricultural and agroindustrial residues, which contribute to the degradation of each area. The sediment contact fish embryo test showed to be powerful tool to detect embryo toxicity in sediments, not only by being a sensitive method, but also for taking into account bioavailability. This test provides an ecological highly realistic and relevant exposure scenario, and should therefore be added in ecotoxicological sediment quality assessments.

Morphology of the female reproductive system of European pea crabs (Crustacea, Decapoda, Brachyura, Pinnotheridae).

Commensal pea crabs inhabiting bivalves have a high reproductive output due to the extension andfecundity of the ovary. We studied the underlying morphology of the female reproductive system in the Pinnotheridae Pinnotheres pisum, Pinnotheres pectunculi and Nepinnotheres pinnotheres using light microscopy and transmission electron microscopy (TEM). Eubrachyura have internal fertilization: the paired vaginas enlarge into storage structures, the spermathecae, which are connected to the ovaries by oviducts. Sperm is stored inside the spermathecae until the oocytes are mature. The oocytes are transported by oviducts into the spermathecae where fertilization takes place. In the investigated pinnotherids, the vagina is of the "concave pattern" (sensu Hartnoll1968): musculature is attached alongside flexible parts of the vagina wall that controls the dimension of its lumen. The genital opening is closed by a muscular mobile operculum. The spermatheca can be divided into two distinct regions by function and morphology. The ventral part includes the connection with vagina and oviduct and is regarded as the zone where fertilization takes place. It is lined with cuticle except where the oviduct enters the spermatheca by the "holocrine transfer tissue." At ovulation, the oocytes have to pass through this multilayered glandular epithelium performing holocrine secretion. The dorsal part of the spermatheca is considered as the main sperm storage area. It is lined by a highly secretory apocrine glandular epithelium. Thus, two different forms of secretion occur in the spermathecae of pinnotherids. The definite role of secretion in sperm storage and fertilization is not yet resolved, but it is notable that structure and function of spermathecal secretion are more complex in pinnotherids, and probably more efficient, than in other brachyuran crabs.

Morphology and function of the reproductive system of representatives of the genus Uca.

The morphology of the reproductive organs of three species of fiddler crabs, Uca ecuadoriensis, Uca c.f. forcipata, and Uca tangeri were investigated to subsequently produce a model of their mode of operation. Vulva, vagina, and spermatheca in females, and the first and second gonopods in males were examined by applying histological techniques and electron microscopy. In all three species, vulva and vagina conform to the concave type, and the spermatheca complies with the ventral type. The tissue of the oviduct orifice is enlarged and bulges into the lumen of the spermatheca. Differences between the three species are apparent in the organization of the spermatheca, especially in the distribution and structure of glandular epithelium: In U. ecuadoriensis and U. c.f. forcipata the largest proportion of the spermathecal wall is lined with cuticle and only a small area consists of glandular epithelium, while in U. tangeri almost all of the lining is glandular. Furthermore, the glandular epithelia of the species differ in their histology and ultrastructure: In U. ecuadoriensis it is tubular and multilayered, while in U. c.f. forcipata it is mono-layered. U. tangeri finally has both forms of this tissue. In the males, the terminal segments of the first gonopod exhibit a tight fit to female organs and narrow, tightly sealed sperm channels. These features suggest a tendency towards minimizing loss of fluids, which can be interpreted as an adaptation to mating on land. The tight fit of male gonopod and female opening seem to be protection from interbreeding, which points toward a strong sexual selection. In the terrestrial environment, these originally aquatic organisms experience serious competition for resources; therefore there is pressure on successful reproduction. According to the current results a model of the process of fertilization and egg-laying involving the investigated organs was generated.

Isolation and phylogenetic analysis of Sindbis viruses from mosquitoes in Germany.

A molecular survey of 16,057 mosquitoes captured in Southwest Germany during the summer of 2009 demonstrated the presence of Sindbis virus (SINV) in Culex spp. and Anopheles maculipennis sensu lato. Phylogenetic analysis of the German SINV strains linked them with Swedish SINV strains, the causative agent of Ockelbo disease in humans.

Changes in toxicity and dioxin-like activity of sediments from the Tietê River (São Paulo, Brazil).

Acute cytotoxicity (neutral red assay), AhR-mediated toxicity (CYP P450 1A induction potential-EROD assay) and concentrations of PAHs were recorded in RTL-W1 cells to assess the ecotoxicological potential of sediments from reservoirs along the Tietê River and the Pinheiros River (Brazil). Almost all sediments tested induced cytotoxicity and stimulated cytochrome P450-associated EROD activity. Toxicity increases from Tietê River spring to São Paulo city region and decreases towards downstream. A closer analysis of chemical measurements of PAHs and results from bioassay revealed that the PAHs analyzed could not explain more than 7% of the EROD-inducing potencies. Results confirm that most of the toxicity is due to the discharges of the metropolitan area of São Paulo. Moreover, they indicate additional sources of pollutants along the river course, which contribute to the degradation of each reservoir.

Sediment genotoxicity in the Tietê River (São Paulo, Brazil): in vitro comet assay versus in situ micronucleus assay studies.

The in vitro comet assay with the permanent fish cell line RTL-W1 and the in situ micronucleus assay using erythrocytes from indigenous tilapia (Oreochromis niloticus) were used to detect genotoxicity in Tietê River sediments (São Paulo, Brazil). Either test was successful in identifying site-specific differences in genotoxicity, with a high correlation between in situ and in vitro results indicating the relevance of the latter even for environmental studies. Discharges from São Paulo city have major impact on genotoxic effects by sediment-bound contaminants; however, overall genotoxicity decreases downstream. The high genotoxic burden of the Tietê River warrants measures to reduce the input of toxic effluents.

Genetic differentiation of populations within the Culex pipiens complex and phylogeny of related species.

The Culex pipiens complex consists of several species, subspecies, forms, races, physiological variants, or biotypes according to different authors and includes the 2 holarctic variants Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus. Differences in morphological characters are overlapping and thus are delimited in their taxonomic value, even when behavioral and reproductive specializations are apparent. Our enzyme electrophoretic study included 7 geographic populations of Cx. pipiens biotype pipiens and 7 of the biotype molestus from several European countries. For comparison, 5 populations of Culex quinquefasciatus from Asia, Africa, and North America were examined. The aim was an assessment of the extent of genetic differences between local populations of the biotypes pipiens and molestus versus the degree of differentiation between geographic populations of both groups. Culex torrentium, Cx. modestus, Culex stigmatosoma, and Culex territans were studied for comparison as taxonomical well-defined species. The population genetic analyses revealed much higher genetic distances between local populations of Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus compared to the low differentiation between geographic populations within each taxon. The UPGMA analysis and F-statistics position the geographic populations in discrete monophyletic clusters. Gene flow between local populations of the biotypes pipiens and molestus could be shown to be lower than gene flow between geographically distant populations within each of the 2 groups, leading to the conclusion that Cx. pipiens biotype molestus could be a distinct taxon. Culex quinquefasciatus could be diagnosed as genetically well separated, in particular by the diagnostic enzyme marker MDH (NADP). Two genetic enzyme markers were identified to differentiate Cx. torrentium from Cx. pipiens s.l. Culex modestus, Cx. stigmatosoma, and Cx. territans showed considerable genetic distances to the species of the Culex pipiens complex and between each other, and several genetic markers could be identified.

Functional morphology of the reproductive system of Galathea intermedia (Decapoda: Anomura).

Spermatophore formation in Galathea intermedia begins in the proximal part of the vas deferens. The contents subsequently form a spermatophoric ribbon, the so-called "secondary spermatophore," in its distal part. A strongly muscular ductus ejaculatorius is present in the coxa of the fifth pereiopod which builds up pressure for the extrusion of the spermatophoric ribbon. After extrusion, the ribbon is caught by the first gonopod, while the second gonopod dissolves the matrix of the ribbon. During copulation the spermatophores are randomly placed on the sternum of the female, near the genital opening, by the fifth pereiopods of the male. Subsequent ovulation of the female via the genital opening, an active process accomplished through muscular activity, results in fertilization of the eggs by the exploding spermatophores. External intersexes are characterized by both male and female external sexual characters, but in all individuals only male gonads are present. No trace of a female reproductive system could be detected. Thus, these external intersexes are exclusively functional males.

Review of the anopheline mosquitoes of Myanmar.

This study examined the species of anopheline mosquitoes in Myanmar. Out of 36 species of anophelines distributed throughout the country, ten species were found to be infected with the malaria parasite. These vectors are presented with particular reference to their distribution and a summary of bionomics and infection rates.

Comparative electron microscopic study of the stomach of Orchestia cavimana and Arcitalitrus sylvaticus (crustacea: Amphipoda).

The functional morphology of stomachs of the European semiterrestrial amphipod Orchestia cavimana and of the Australian terrestrial species Arcitalitrus sylvaticus was studied by electron microscopy. The stomach of the two amphipod species is divided longitudinally into a spacious dorsal food channel and two ventral filtration channels. Additionally, a prominent helically oriented circulation channel is situated on each lateral side of the stomach, forming a semicircular channel separated from the food channel by spines. The food channel conveys coarse food particles directly into the midgut through a funnel. The filtration channels receive fine material filtered through primary and secondary filters. Material forced through the secondary filters by the pressure of the laterally located inferolateralia eventually reaches the openings of the midgut glands. Washing of filters and soaking of ingested food items with enzymes probably is achieved by a forward stream of digestive juice from the midgut glands and conveyed through the circulatory channels. The specializations of the stomach of the two species of Amphipoda investigated are described and compared to the pertinent structures of Mysidacea and Isopoda.

Molecular and electron microscopic identification of Wolbachia in Culex pipiens complex populations from the Upper Rhine Valley, Germany, and Cebu City, Philippines.

We detected Wolbachia infections in Culex pipiens complex mosquitoes from the Upper Rhine Valley, Germany, and Cebu City, Philippines, with the use of polymerase chain reaction (PCR) amplification of the 16S rDNA of the bacteria and further confirmation by electron microscopy. Species of 5 different autogenous and anautogenous populations of Culex pipiens from Germany and 1 population of Culex quinquefasciatus from the Philippines were used in this study. Larvae were sampled from different localities and reared to the adult stage for further processing. Whole adult males and the heads of adult females were processed by PCR, whereas the abdomens of females were used for electron microscopic study when an infection was detected by PCR. All Culex populations assayed by PCR showed infection of Wolbachia at rates between 10 and 100%. Females from different populations exhibited higher infection rates than did the males, and, likewise, Cx. pipiens populations that were autogenous showed higher infection rates of Wolbachia than did anautogenous individuals. In Cx. quinquefasciatus populations, males were infected at 33.33% and females, only 10%. We studied the ultrastructure of Wolbachia in the ovaries of species belonging to Cx. pipiens complex by means of transmission electron microscopy. The bacteria exhibited typical morphology for Wolbachia with 3 enveloping membranes.

Anopheles dirus and its role in malaria transmission in Myanmar.

Anopheles dirus is one of the primary vectors of highly drug-resistant Plasmodium falciparum, which causes cerebral malaria resulting in high mortality. Mosquito collections were conducted in a forest wood-extraction area (Bago Division), an irrigated plain area near foothills (Mandalay Division), a coastal plain (from domestic wells in the Mudon area, Mon State) near the foothill area, as well as a hilly area (deep forest timber extraction camp, Tanintharyi Division) from May 1998 to March 2000. This study examined adult bionomics of An. dirus and its relationship to malaria transmission as an aid in the control of malaria in different ecological settings in these particular regions. Within these areas, Mudon, Mon State, has a high incidence of malaria. To investigate this malaria, blood smear examinations were conducted among the local people in Mudon, Mon State. During the study period, malaria blood smear slide-positive rates ranged between 9.9% and 34.28% throughout the year. The ultimate goal of these studies was to help in formulating an improved malaria control program involving microbial control agents in this area.

Studies on the bionomics of Anopheles dirus (Culicidae: Diptera) in Mudon, Mon State, Myanmar.

This study examined some environmental factors influencing the larval habitats of Anopheles dirus (breeding in wells) in Mudon, Myanmar, from May 1998 to March 2000. The larval/pupal density was found to be directly proportional to rainfall and indirectly proportional to the well water level. Shade, vegetation and debris on the surface of well water were important factors influencing the abundance of the aquatic stages of An. dirus. Salinity had an inverse correlation with the larval and pupal density. Other mosquito species associated with An. dirus were identified. Important predators of the mosquito larvae were larvivorous fish, damselfly and dragonfly nymphs. All wells examined were lined with lateritic rocks. Chemical analysis of water samples from wells was conducted.

Morphology and function of the copulatory system in freshwater crabs of the genus Potamon.

To understand the reproductive processes of freshwater crabs of the genus Potamon, we examined the first and second gonopod and the gonoducts of the female by histological methods. The gonopods are highly modified compared to those of other brachyuran crabs. In particular, the second gonopod is unusually long and has a special morphology, ending in a long sclerotized tube. Suggestions for the function of both gonopods and their different parts are presented. Tubulation of the first and second gonopod is observed. Rosette glands, which are abundant in the subterminal joint of the first gonopod, are connected to the sperm channel by cuticular pores. In females, the chitinous parts of the inner vulva may prove to have a more constant morphology than the external flexible structures. J. Morphol. 239:157-166, 1999. © 1999 Wiley-Liss, Inc.

The impact of lead on the assimilation efficiency of laboratory-held Diplopoda (Arthropoda) preconditioned in different environmental situations.

In five diplopod species from three families collected from locations with different soil metal contents, the following parameters were examined: the litter mass (and energy) ingested per day, the mass (and energy) assimilation rates, and the litter mass (and energy) assimilated per day, when the diplopods were fed (1) uncontaminated and (2) artificially lead-enriched leaf litter. These parameters were compared between species and between animals from different sites. The mass and energy assimilation efficiencies depend on (1) the size of the species and (2) the collection site of the animals. Relative mass (and in three of the species examined also energy) assimilation was highest in those animals collected from the less contaminated sites. With artificial diets, specimens from a site with soil contaminated by heavy metals showed greater assimilation of both mass and energy than originally unaffected specimens. One species (Glomeris conspersa) was able to compensate for the low assimilation rate by increased consumption, and thus guarantee a sufficient energy supply. One of the other species (Polydesmus denticulatus), however, did not show this compensation. Animals of this species from less contaminated sites showed a very low absolute energy assimilation rate and high mortality under lead treatment.

Scanning and transmission electron microscopic observations on the stomach of three mysid species (Crustacea).

Epithelial and cuticular linings of the stomach investigated in three species representing different genera of the Mysidacea are elaborated into a set of structural specializations dividing the stomach longitudinally into one dorsal and two ventral channels. The dorsal, or food, channel contains ingested food and retains coarse particles, which eventually are transported into the midgut through a funnel. The ventral, or filtration, channels, which are separated by an anterior and a posterior median ridge (anteromedianum, inferomedianum), contain fine particles and soluble materials extracted from the dorsal channel through two filter systems: primary filters, which lie anteriorly on either side of the anteromedianum, and posterior secondary filters, which are located on the inferomedianum. The final filtrate is transported into the ventral caeca or midgut glands. The ultrastructure of the cuticle lining the lumen of the stomach shows several specializations, the most prominent of which are stout spines and delicate filter devices. The epithelium is multilayered in circumscribed areas (the lateralia). The basement lamina is extremely developed in the inferomedianum. Detailed knowledge of the microscopic anatomy and the ultrastructure of the stomach allows identification of several homologous gastric structures among different peracaridean groups and in Decapoda.