All nymphal instars of the recently discovered troglobitic planthopper species Valenciolenda fadaforesta Hoch & Sendra, 2021 are described. This represents the first documentation of the complete postembryonic development of any species in the family Kinnaridae. Characters of the external morphology are described and illustrated, and a key to the instars are provided to facilitate discrimination among the different nymphal stages. While V. fadaforesta nymphs share certain synapomorphies with other Fulgoromorpha (except the Tettigometridae), e.g., the cog-wheel structures of the metatrochanters, other characters may be correlated with the subterranean way of life of the species, and thus be autapomorphic, such as the absence of compound eyes in all instars.

Caves, Iberian Peninsula, morphology, taxonomy, troglobite

Fulgoromorpha is a large subgroup of the Hemiptera comprising at least 14,000 species in 21 extant and 15 extinct families (Bourgoin 2022). Despite their worldwide distribution (Bourgoin 2022), ecological diversity and economic importance (Denno and Perfect 1994), and many aspects of their biology are scarcely known. While postembryonic development has been studied in more detail in select Fulgoromorpha taxa (see e.g., Yang and Yeh 1994, and references therein, Emeljanov 1996; Asche 2015), information on nymphal morphology remains incomplete or lacking for most species. The only comprehensive treatments of Fulgoromorpha nymphs to date, including representatives of most taxa in family rank to date has been provided by Yang and Yeh (1994) and Emeljanov (2001). From their observations Yang and Yeh (1994) derived a compilation of characters considered to be distinctive of the 5^th instar of Fulgoromorpha nymphs, and Emeljanov provided a hypothetical ground plan of certain aspects of nymphal morphology of Fulgoromorpha. One of the taxa which were neither covered by Yang and Yeh (1994) nor Emeljanov (2001) are the Kinnaridae. This family currently consists of 116 described species across 25 genera from the Palearctic, East Africa (partim), Arabian Peninsula and Oriental regions, as well as from the Nearctic and the Neotropics (Bourgoin 2022).

The nymphs of Kinnaridae have until recently been entirely unknown. This is most likely due to the lack of material, the reason for which may lie in their cryptic way of life. These apparently live in or close to the soil and/or subterranean crevices presumably feeding on roots (Fennah 1948, 1980). The nymphs of epigean species supposedly leave the nymphal habitat before or after the adult molt and feed and reproduce above ground, while adults of troglobitic species complete their life cycle underground. Cave-dwelling species of Kinnaridae are documented from Mexico (Fennah 1973), Jamaica (Fennah 1980), Brazil (Hoch and Ferreira 2013, 2016) and Spain (Hoch et al. 2021).

The first description of a kinnarid nymph (instar V) was given by Hoch et al. (2021) for the troglobitic Valenciolenda fadaforesta Hoch & Sendra, 2021 from Spain. Described from a cave in eastern karstic massif in the east of Iberian Mountain Range, in Valencia, Spain (the type locality), adults and nymphs were found to co-occur in the same habitat, thus offering the opportunity to obtain and study nymphs of all instars (Fig. 1).

Figure 1. 

Valenciolenda fadaforesta, habitus of adult male (left) and instar nymph V (right). Body length of adult: 3.6 mm; body length of nymph: 2.9 mm. (Photo courtesy by Roberto García-Roa, www.robertogarciaroa.com).

Here we provide the first descriptions of the external morphology of instars I–IV of a representative of the Kinnaridae and summarize the results of Hoch et al. (2021). Our observations contribute to the knowledge of morphological alteration during nymphal development and help to complete the ground pattern of nymphal instars in Fulgoromorpha.

The observable changes between the different nymphal stages of Valenciolenda fadaforesta consist of an increase in body size, an increase in the number of sensory pits and wax-secreting pores present in both the thorax and the abdomen, and the development of wing pads that develop into wings once they have reached maturity. The increase in length of the metathoracic tibiae and femora, as well as in the number of sensory pits and wax-secreting pores present in the tergites of the thoracic segments, have been the diagnostic characters used for the separation and classification of the different nymphal stages. The structures studied in the cuticle of V. fadaforesta consist of sensory organs composed by wax filaments located in small pits, distributed throughout the thoracic region and in the caudal segments of the abdominal region, as well as small wax-secreting pores located in the postnotum of the metanotum. These individuals secreted a waxy substance that filled the cavities of the cuticle, made it difficult to study the structures and hide the pits where the wax filaments are located. So, it is possible that some of the smaller pits have gone unnoticed. The clear visibility of the wax-secreting pores present in the metathoracic postnotum has been very useful to differentiate between the smaller nymphal stages. It should be noted, however, that there exists some interindividual and intraindividual variability, with specimens that present a variable number of wax-secreting pores on both sides of the thorax (Fig. 5).

Figure 5. 

Nymphal instar III of Valenciolenda fadaforesta (SEM). Thorax in dorsal view A specimen 1 B specimen 2. Note variation among individuals. In specimen 1 a waxy substance covers the tergites, absent in the specimen 2. The variation in the number of structures present in the same organism can also be observed in specimen 2, with 7 pores in the left section and 8 on the right.

The life cycle of this species is unknown. Currently, we have no recorded evidence on the duration of single instars, overall lifespan, copulation, oviposition, or the eggs themselves. Furthermore, the literature on nymphal morphology and its development in the Kinnaridae family is also lacking, and the taxon has not been examined in the studies on nymphal morphology of the fulgoromorphs by Yang and Yeh (1994) and Emeljanov (2001) due to the lack of material.

The life cycle of V. fadaforesta is an hemimetabolous cycle composed of five nymphal stages and the adult stage. Juvenile stages of hemimetabolous insects resemble adults, except for the absence of functional wings and immature genital configuration. In these life cycles, embryogenesis gives rise to nymphs that possess adult characteristics. Development of these nymphs consists of growth in size during which the growth ratio of the linear dimensions of the exoskeleton tends to remain constant between nymph-to-nymph molts (Bellés 2020). Nymphs gradually develop into adult characteristics, with the final moult bringing forth the adult which has functional wings and fully developed external genitalia.

In V. fadaforesta the growth and development of the nymphal stages seems to follow a linear progression according to what can be observed (see Fig. 2). The increase of overall body length and proportions of femora/tibia length is more or less gradual from instar I – IV, while the change from instar IV –V is notably more distinct. Although we attempted to collect individuals across all possible sizes, these were small insects with tiny variations in size between different nymphal stages, which were difficult to discern with the naked eye. Apparently, overall body length alone is not sufficient to discriminate the nymphal stages, as it notably varies among individuals of the same instar. The observed variation may be due to genetic variation or may be due to individual modification during ontogeny (freshly hatched nymphs of a given instar are likely to be smaller than nymphs closer to the consecutive molt), or even caused by physiological condition (e.g., starved vs. well-fed). The cuticle in nymphs is not fully hardened, even in sclerites, and especially intersegmental membranes are rather extensible. The correct nymphal stage can ultimately only be determined upon examination of cuticle structures and measurement of the legs. The base pattern of the fulgoromorphan Hemiptera consists of five nymphal stages, with the only exception of Lycorma delicatula (White, 1845) (Fulgoridae) which has only four (Dara et al. 2015). Our observations on the nymphal development of V. fadaforesta are consistent with the existing literature on this Hemiptera taxon (Liang 2001; Hoch and Ferreira 2016; Hoch et al. 2021) and other closely related taxa (Wilson and Tsai 1982; Chen and Yang 1995; McPherson and Wilson 1995).

In the samples examined, individuals belonging to the same nymphal stage can be observed showing great differences in the body size and size of the abdominal region. When observed under a stereomicroscope, some individuals had a more “swollen” abdomen, although the size of the thorax and the cuticle that covers the abdominal tergites maintained its proportions (Fig. 6). At first glance, this variation in the size of the abdomen could result in a misclassification of the nymphal stages. Thus, measurement of its legs and the confirmation of key structures in its cuticles (i.e., presence or absence of wing pads, relative size of wing pads, number of sensory plate organs on the pedicel, number and distribution of sensory pit organs on pronotum, mesonotum and metanotum and number and distribution of wax-secreting pores) are required.

Figure 6. 

Body size variation in nymphal instars I–V (stereomicroscope photograph). Nymphal instar V are largest but display high degree of variation in overall abdomen size (see text for possible explanation).

Regarding the size of nymphs, there was some variation of overall body size among individuals of the same nymphal instar, perhaps due to physiological conditions and individual development. Phenotypic variation in growth rates was present in all living organisms, and their plasticity was affected by multiple environmental variables where temperature and food availability were most influential (Lee and Roh 2010). Regarding temperature, underground habitats are one of the few natural systems where microclimatic conditions can reach levels of homogeneity otherwise only achieved under laboratory conditions. The stable conditions in the deep cave zone thus rule out temperature oscillations as the determining factor underlying the observed phenotypic variation among individual nymphs of the same instar, e.g., in total body size (Sánchez-Fernández et al. 2018). The section of the cave where most of the nymphs were collected, the so-called “Root Room” (or Sala de las Raíces) in Murciélagos Cave, is no exception, with little variable temperatures between 16.5 °C and 18.3 °C throughout the year (Sendra et al. 2015). The variations in the size of the collected individuals could then be explained by differences in the stage of development or by differential access to the food source.

The gradual increase of absolute and especially relative length of the hind-tibia in consecutive instars may be indicative of increasing mobility in the course of nymphal development. Field observations of another obligately cavernicolous planthopper species, although in a different taxon of family rank, Oliarus polyphemus (Cixiidae) from lava tubes on Hawaii Island, revealed that earlier instars (I–III/IV) appear to be rather sedentary, sitting on roots and feeding. Older instars however, especially instar V, show dynamic mobility patterns, actively moving away from roots, most likely in search of a secure place suitable for molting into adults (Hoch, personal observation; also see Hoch and Howarth 1993). It is conceivable that nymphs of Valenciolenda fadaforesta show similar mobility patterns.

To discern the sexes of immatures is challenging. Nymphal instars I–IV do not present sexual structures that allow clear differentiation between male and female specimens. In the fifth nymphal stage, two different genital phenotypes exist: in some, yet not all, V instar nymphs we found two inconspicuous conical processes in the posteroventral region of segment IX (Fig. 7). These were only detectable under high magnification (SEM), and could neither be seen with the naked eye or even with the use of a stereomicroscope. These conical processes have been interpreted by Hoch et al. (2021) to be either precursors of the genital styles of the male or the gonocoxae VIII of the female. Sexing immatures of Valenciolenda fadaforesta is further impeded by the fact that highly modified external female genitalia are characteristic for the Kinnaridae (as well as the closely related taxon Meenoplidae), and even females of epigean species display strongly reduced ovipositors (Wilson 1983).

Figure 7. 

Nymphal instar V of Valenciolenda fadaforesta (SEM). Tip of abdomen in dorsodistal view. Conical processes (A, arrow) located medially at the level of the posterioventral corner of tergite IX present in A and absent in B.

The results and observations presented here are based exclusively on field samples. These first ever descriptions of all nymphal instars of a species of the Kinnaridae add to the existing knowledge of postembryonic development in this taxon, and further contribute to the reconstruction of ground pattern, or body plan, of Fulgoromorpha nymphs. It should be kept in mind, however, that the study organism, Valenciolenda fadaforesta, is an obligate troglobiont, and thus a highly specialized species. Certain morphological configurations may have evolved as reductive characters and may thus represent autapomorphies for this particular species rather than for the entire family. Since Valenciolenda fadaforesta is the only representative of the Kinnaridae of which nymphs have been available for study, a comparison with nymphs of epigean Kinnaridae species has not been possible. Accordingly, we cannot with certainty distinguish between potential autapomorphies for the species and those for the entire lineage. Suspected autapomorphies for V. fadaforesta are the absence of compound eyes in all instars, and perhaps related to it, the extremely short and wide vertex. Similar alterations of head capsule architecture have been observed in other cavernicolous Fulgoromorpha. Hoch and Howarth (1989), in a study on cavernicolous Cixiidae from Australia, hypothesized that “the broadening of the vertex [...] is apparently correlated with the reduction of compound eyes, and might have resulted from mechanical competition within the head capsule during morphogenesis, analogous to that which Berger and Howard (1968) reported for birds”.

Given its occurrence in a specialized habitat yet in a comparatively easily accessible location, in the vicinity of academic facilities, Valenciolenda fadaforesta is a potential model organism to study morphogenesis in an obligately cavernicolous insect. In order to develop a protocol for rearing Valenciolenda fadaforesta in the laboratory, further studies are required to understand life history, longevity, reproduction rate, and host associations as well as to determine environmental factors controlling population size. Information on these will also be pivotal in developing strategies for management and conservation of the caves and other organisms inhabiting them.

We would like to express our sincere thanks to Loles Beltrán, Santiago Teruel, Agustín Moreno, and Mireia & Raul López who helped us during the visits of the Murciélagos Cave and collecting specimens. We also thank our colleagues at the Servicio Central de Apoyo a la Investigación Experimental (SCSIE) and the Universitat de València for allowing us the use of their SEM facilities. We would like to express our thanks to Roberto García-Roa for allowing us to use his magnificent photographs of the specimens.